Glp-1 receptor agonist compounds with a modified n-terminus

ABSTRACT

The invention relates to GLP-1 receptor agonist compounds with a modified N-terminus. The compounds are of the formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1 receptor agonist peptide lacking the two N-terminal amino acid residues; and Y—Z represents novel His-Ala mimetics. Examples of GLP-1 receptor agonist compounds are derived from human GLP-1 (7-37), exendin-4(1-39), or GLP-1 A (1-37). The invention also relates to derivatives of these compounds, in particular compounds with one or more albumin binding side chains capable of protracting the duration of action in vivo of these compounds. The peptides and derivatives of the invention have a good potency, a protracted pharmacokinetic profile, are stable against degradation by gastro intestinal enzymes, and/or have a high oral bioavailability. These properties are of importance in the development of GLP-1 receptor agonist compounds for subcutaneous, intravenous, and/or in particular oral administration. The invention also relates to intermediate products for use in the preparation of the GLP-1 receptor agonist compounds of the invention.

FIELD OF THE INVENTION

The present invention relates to analogues and derivatives of GLP-1receptor agonist peptides, and their pharmaceutical use. In the GLP-1receptor agonist peptides of the invention, such as Glucagon-LikePeptide-1 (GLP-1), exendins and analogues thereof, the two N-terminalamino acids have been replaced by N-terminal mimetics.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The Sequence Listing, entitled “SEQUENCE LISTING”, is 1770 bytes, wascreated on 1 Dec. 2010, and is incorporated herein by reference.

BACKGROUND OF THE INVENTION

WO 2004/067548 A2 relates to chemically modified metabolites ofregulatory peptides and methods of producing and using same.

Liraglutide, a GLP-1 derivative for once daily administration which ismarketed by Novo Nordisk A/S, is disclosed in Example 37 of WO 98/08871.

Semaglutide, a GLP-1 derivative for once weekly administration which isunder development by Novo Nordisk A/S, is disclosed in Example 4 of WO06/097537.

SUMMARY OF THE INVENTION

The invention relates to GLP-1 receptor agonist compounds comprising amodified N-terminus.

Preferred compounds have the formula Chem. 1: Y—Z—P, wherein Prepresents a fragment of a GLP-1 receptor agonist peptide lacking theN-terminus; and Y—Z represents a group mimicking the N-terminus of thepeptide. The new N-terminal is preferably a His-Ala, a His-Gly, and/or aHis-Ser mimetic.

More in particular the invention relates to a GLP-1 receptor agonistpeptide having the formula Chem. 1: Y—Z—P, wherein P represents afragment of a GLP-1 receptor agonist peptide lacking the two N-terminalamino acid residues; Z represents a group of the formula Chem. 2:

wherein W represents a group of formula Chem. 3:

wherein R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl,heterocyclyl, or heteroaryl; and Y represents a group of formula Chem.4:

or Chem. 5:

wherein X₁ is N, O, or S; X₂, X₃, X₄, and X₅ independently represent C,or N, with the proviso that at least one of X₂, X₃, X₄ and X₅ is C; R11,R12, R13, and R14 independently represent hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl; Q represents a bond, or a group of formula

*—(C(R15)(R16))_(q)—*,  Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other andindependently for each value of q represent hydrogen, alkyl, carboxyl,or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

The invention also relates to a derivative of this peptide, and apharmaceutically acceptable salt, amide, or ester thereof.

The invention also relates to the pharmaceutical use of these compounds,preferably for the treatment and/or prevention of all forms of diabetesand related diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.

Finally, the invention relates to intermediate products corresponding tothe new N-terminus, as well as to the peptide fragments, i.e. beforeattachment of the new N-terminus, both relevant for the preparation ofthe peptides of the invention.

The peptides and derivatives of the invention are biologically active,preferably of a high potency. Also, or alternatively, they have aprotracted pharmacokinetic profile. Also, or alternatively, they arestable against degradation by gastro intestinal enzymes. Also, oralternatively, they have a high oral bioavailability. These propertiesare of importance in the development of next generation GLP-1 compoundsfor subcutaneous, intravenous, and/or in particular oral administration.

DESCRIPTION OF THE INVENTION

The invention relates to a GLP-1 receptor agonist peptide having theformula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1receptor agonist peptide lacking the two N-terminal amino acid residues;Z represents a group of the formula Chem. 2:

wherein W represents a group of formula Chem. 3:

wherein

R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl,heterocyclyl, or heteroaryl; and Y represents a group of formula Chem.4:

or Chem. 5:

wherein X₁ is N, O, or S; X₂, X₃, X₄, and X₅ independently represent C,or N, with the proviso that at least one of X₂, X₃, X₄ and X₅ is C; R11,R12, R13, and R14 independently represent hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl; Q represents a bond, or a group of formula

*—(C(R15)(R16))_(q)—*,  Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other andindependently for each value of q represent hydrogen, alkyl, carboxyl,or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

In a first aspect, R1 and R2 do not both represent hydrogen, and theinvention accordingly relates to a GLP-1 receptor agonist peptide havingthe formula Chem. 1: Y—Z—P, wherein P represents a fragment of a GLP-1receptor agonist peptide lacking the two N-terminal amino acid residues;Z represents a group of the formula Chem. 2:

wherein W represents a group of formula Chem. 3:

wherein R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl,heterocyclyl, or heteroaryl, with the proviso that (iii) R1 and R2 donot both represent hydrogen; and Y represents a group of formula Chem.4:

or Chem. 5:

wherein X₁ is N, O, or S; X₂, X₃, X₄, and X₅ independently represent C,or N, with the proviso that at least one of X₂, X₃, X₄ and X₅ is C; R11,R12, R13, and R14 independently represent hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl; Q represents a bond, or a group of formula

*—(C(R15)(R16))_(q)—*,  Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other andindependently for each value of q represent hydrogen, alkyl, carboxyl,or hydroxyl; and R represents hydrogen, or alkyl; or a pharmaceuticallyacceptable salt, amide, or ester thereof.

In a second aspect, R1 and R2 may both represent hydrogen, and Q-NR—* isnot attached to a nitrogen atom of Chem. 4.

In a third aspect, R1 and R2 may both represent hydrogen, and Q-NR—* isattached to a carbon atom of Chem. 4.

The invention also relates to a derivative of each of these peptides,and to pharmaceutically acceptable salts, amides, or esters thereof.

The invention also relates to the pharmaceutical use of these compounds,preferably for the treatment and/or prevention of all forms of diabetesand related diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.

Finally, the invention relates to intermediate products corresponding tothe new N-terminus, as well as to the peptide fragments, i.e. beforeattachment of the new N-terminus, both relevant for the preparation ofthe peptides of the invention.

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ε=epsilon;γ=gamma; ω=omega; etc. Also, the Greek letter of μ may be represented by“u”, e.g. in μl=ul, or in μM=uM.

An asterisk (*) in a chemical formula designates i) a point ofattachment, ii) a radical, and/or iii) an unshared electron.

GLP-1 Receptor Agonist

The GLP-1 receptor agonist compounds of the invention may be derived, orare derivable, from human GLP-1(7-37), exendin-4(1-39), and/orGLP-1A(1-37). The amino acid sequences of these peptides may be found inthe UniProt Knowledgebase (UniProtKB)—SwissProt section(www.uniprot.org) with the following accession numbers, sequenceidentifiers, and sequence names: UNIPROT:P01275_(—)8, GLUC_HUMAN,Glucagon-like peptide 1(7-37); UNIPROT:P26349_(—)3, EXE4_HELSU,Exendin-4, or exenatide; and UNIPROT:042143_(—)5, GLUC1_XENLA,Glucagon-like peptide 1A; respectively.

The sequences of the corresponding fragments lacking the two N-terminalamino acids, viz. GLP-1(9-37), exendin-4(3-39), and GLP-1A(3-37), areincluded in the appended sequence listing as SEQ ID NO: 1, SEQ ID NO: 2,and SEQ ID NO: 3, respectively.

Another example of a GLP-1 receptor agonist fragment from which thecompounds of the invention may be derived, or are derivable, is thepeptide designated exendin-3(3-39) which is the D3 analogue of SEQ IDNO: 2, i.e. identical to SEQ ID NO: 2 except for having aspartic acid(D, Asp) at position 3, the first amino acid residue.

The sequence of the GLP-1 receptor may be found in the UniprotKBdatabase referred to above with the following accession number,identifier, and name: UNIPROT:P43220, GLP1R_HUMAN, Glucagon-like peptide1 receptor, GLP-1 receptor, GLP-1-R, or GLP-1R.

The term “GLP-1 receptor agonist” as used herein refers to a compoundwhich is an agonist of the human GLP-1 receptor, i.e. a compound thatstimulates the formation of cAMP in a medium containing the human GLP-1receptor. GLP-1 receptor agonism, or potency, is determined as describedbelow, in the section headed “Potency”, see also Example 13 herein.

Amino Acids and Peptides

The term “peptide”, as e.g. used in the context of the GLP-1 receptoragonist peptides of the invention, refers to a compound which comprisesa series of amino acids intereconnected by amide (or peptide) bonds.

In a particular embodiment the peptide is to a large extent, orpredominantly, composed of amino acids interconnected by amide bonds(e.g., at least 50%, 60%, 70%, 80%, or at least 90%, by molar mass). Inanother particular embodiment the peptide consists of amino acidsinterconnected by peptide bonds.

The peptides of the invention comprise at least five constituent aminoacids connected by peptide bonds. In particular embodiments the peptidecomprises at least 10, preferably at least 15, more preferably at least20, even more preferably at least 25, or most preferably at least 28amino acids.

In particular embodiments, the peptide is composed of at least fiveconstituent amino acids, preferably composed of at least 10, at least15, at least 20, at least 25, or most preferably composed of at least 28amino acids.

In additional particular embodiments, the peptide is a) composed of, orb) consists of, i) 29, ii) 30, iii) 31, or iv) 32 amino acids.

In still further particular embodiments, the peptide is a) composed of,or b) consists of, i) 33, ii) 34, iii) 35, or iv) 36 amino acids.

In a still further particular embodiment the peptide consists of aminoacids interconnected by peptide bonds.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes proteogenic amino acids (encoded by thegenetic code, including natural amino acids, and standard amino acids),as well as non-proteogenic (not found in proteins, and/or not coded forin the standard genetic code), and synthetic amino acids. Thus, theamino acids may be selected from the group of proteinogenic amino acids,non-proteinogenic amino acids, and/or synthetic amino acids.

Non-limiting examples of amino acids which are not encoded by thegenetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.Non-limiting examples of synthetic amino acids are the D-isomers of theamino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyricacid), β-alanine, and des-amino-histidine (desH, alternative nameimidazopropionic acid, abbreviated Imp).

In what follows, all amino acids for which the optical isomer is notstated is to be understood to mean the L-isomer (unless otherwisespecified).

GLP-1 Receptor Agonist Peptides, Fragments, Analogues, ResidueNumbering, Identity

A “GLP-1 receptor agonist peptide” is a peptide as defined above, andalso a GLP-1 receptor agonist as defined above.

The peptides of the invention are GLP-1 receptor agonist peptides.

Additional examples of GLP-1 receptor agonist peptides are the followingknown compounds: Human GLP-1(7-37), exendin-4(1-39), exendin-3(1-39),and GLP-1A(1-37).

In a particular embodiment, the GLP-1 receptor agonist compound of theinvention may be derived, or is derivable, from any one or more of theseknown GLP-1 receptor agonist peptides.

The term “fragment” as it refers to a GLP-1 receptor agonist peptidemeans a peptide which is shorter than the peptide referred to.

In a particular embodiment corresponding to the definition of group P informula I, the fragment lacks the two N-terminal amino acids as comparedto the corresponding full-length peptide being a GLP-1 receptor agonist.

In another particular embodiment this particular fragment is not initself a GLP-1 receptor agonist, due to a i) substantial, ii) preferablyalmost complete, or iii) more preferably for all practical purposescomplete, loss of biological activity (i.e., GLP-1 receptor agonism).

Particular examples of P (fragments of a GLP-1 receptor agonist peptidelacking the two N-terminal amino acid residues) are the following:GLP-1(9-37), exendin-4(3-39), and GLP-1A(3-37), which are included inthe appended sequence listing as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ IDNO: 3, respectively. Another example of P is exendin-3(3-39) which isvariant D3 of SEQ ID NO: 2.

In the sequence listing, the first amino acid residue of the fragment ofGLP-1(9-37) (SEQ ID NO: 1), which is glutamic acid, is assigned no. 1.However, in what follows—according to established practice in theart—this glutamic acid residue is referred to as no. 9, and subsequentamino acid residues are numbered accordingly, ending with glycine no.37.

Likewise, in the sequence listing, the first amino acid residue ofexendin-4(3-39) (SEQ ID NO: 2), which is also glutamic acid, is assignedno. 1. However, in what follows—according to established practice in theart—this glutamic acid residue is referred to as no. 3, and subsequentamino acid residues are numbered accordingly, ending with serine as no.39.

Likewise, in the sequence listing, the first amino acid residue ofGLP-1A(3-37) (SEQ ID NO: 3), which is aspartic acid, is assigned no. 1.However, in what follows—according to established practice in theart—this aspartic acid residue is referred to as no. 3, and subsequentamino acid residues are numbered accordingly, ending with serine as no.37.

Therefore, generally, any reference herein to an amino acid residuenumber or a position number in the context of the peptides of SEQ ID NO:1, 2, or 3 or analogues thereof is to the sequence starting with Glu atposition 9, Glu at position 3, or Asp at position 3, respectively; andending with Gly at position 37, Ser at position 39, or Ser at pos. 37,respectively.

Additional particular examples of P are analogues of SEQ ID NO: 1, SEQID NO: 2, and/or SEQ ID NO: 3.

An “analogue” as used herein in the context of SEQ ID NO: 1, 2, or 3refers to a peptide, or a compound, which is a variant of any one ormore of SEQ ID NO: 1, 2, or 3.

In a particular embodiment, the analogue of SEQ ID NO: 1 refers to amodification of SEQ ID NO: 1 in which a number of amino acid residueshave been exchanged as compared to SEQ ID NO: 1. These exchanges, ormodifications, may represent, independently, one or more amino acidsubstitutions, additions, and/or deletions. Additions at the N-terminusare, however, preferably excluded. Analogues of SEQ ID NO: 2, and SEQ IDNO: 3 are defined similarly, by analogy to the definition of analoguesof SEQ ID NO: 1.

Analogues may be described by reference to a reference sequence, thenumber of the amino acid residue in the reference sequence correspondingto the one which is modified, i.e., its position, and to the actualmodification.

In particular embodiments, the reference sequence is i) GLP-1(9-37) (SEQID NO: 1); ii) exendin-4(3-39) (SEQ ID NO: 2); or iii) GLP-1A(3-37) (SEQID NO: 3).

The following are non-limiting, illustrative examples of suitableanalogue nomenclature, as used herein:

N⁹-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Lys¹⁸,Glu²²,Gln³⁴]GLP-1(9-37)peptide is a GLP-1 receptor agonist peptide of the invention derivablefrom GLP-1(9-37) (SEQ ID NO: 1), i.e. P in Chem. 1 is an analogue of SEQID NO: 1, viz. the analogue in which the serine at position 18 has beensubstituted with lysine, the glycine at position 22 has been substitutedwith glutamic acid, and the lysine at position 34 has been substitutedwith glutamine;

N⁹-{2-[2-(1H-Imidazol-4-yl)propylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)Glu³⁸-peptideis a GLP-1 receptor agonist peptide of the invention derivable fromGLP-1(9-37) (SEQ ID NO: 1), i.e. P in Chem. 1 is an analogue of SEQ IDNO: 1, viz. the analogue in which the alanine at position 30 has beensubstituted with glutamic acid, the arginine at position 36 has beensubstituted with lysine, and a glutamic acid has been added at theC-terminus, viz. at position 38; and

N³-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Arg¹⁷,Arg²⁰,Arg³³,Lys³⁸]GLP-1A(3-37)-peptideis a GLP-1 receptor agonist peptide of the invention derivable fromGLP-1A(3-37) (SEQ ID NO: 3), i.e. P in Chem. 1 is an analogue of SEQ IDNO: 3, viz. the analogue in which the lysines at position 17, 20, and 33have been substituted with arginine, and a lysine has been added at theC-terminus, viz. at position 38.

This peptide, by the way, is also derivable from SEQ ID NO: 1, and itcan therefore also be designated as analogue (17T, 18Q, 19Q, 21D, 22E,23R, 26R, 30D, 33I, 34N, 36G, 37P, 38S, 39R, 40E, 41I, 42I, 43S, 44K) ofGLP-1(9-37) (SEQ ID NO: 1), having{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}- attachedto the N-terminus, N⁹.

As another example, a GLP-1 receptor agonist peptide of the inventionwhich “comprises at least one of the following substitutions as comparedto GLP-1(9-37) (SEQ ID NO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K; 37K;and/or 38E” refers to a GLP-1 receptor agonist peptide in which P ofChem. 1 is considered an analogue of SEQ ID NO: 1, which analogue has alysine at position 18, a glutamic acid at position 22, a glutamic acidat position 30, a histidine at position 31, a glutamine at position 34,a lysine at position 36, a lysine at position 37, and/or a glutamic acidat position 38, and which analogue may comprise further modifications ascompared to SEQ ID NO: 1.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent.

The expressions “a position equivalent to” or “corresponding position”may be used to characterise the site of modification in a modified GLP-1receptor agonist peptide sequence by reference to any one or more of SEQID NO: 1, 2, or 3. Equivalent or corresponding positions, as well as thenumber of modifications, are easily deduced, e.g. by simple handwritingand eyeballing; and/or a standard protein or peptide alignment programmay be used, such as “align” which is a Needleman-Wunsch alignment. Thealgorithm is described in Needleman, S. B. and Wunsch, C. D., (1970),Journal of Molecular Biology, 48: 443-453, and the align program byMyers and W. Miller in “Optimal Alignments in Linear Space” CABIOS(computer applications in the biosciences) (1988) 4:11-17. For thealignment, the default scoring matrix BLOSUM50 and the default identitymatrix may be used, and the penalty for the first residue in a gap maybe set at −12, or preferably at −10, and the penalties for additionalresidues in a gap at −2, or preferably at −0.5.

This algorithm may also suitably be used for determining the degree ofidentity of the P-group of a GLP-1 receptor agonist peptide of theinvention to each of SEQ ID NO: 1, 2, and 3, e.g. with a view todetermining which of these three sequences has the highest percentage ofidentity to the P-group in question, and thus for determining the numberof amino acid residues that have been exchanged as compared to theclosest related sequence of SEQ ID NOs: 1-3 (the one with the highestpercentage of identity). If the percentages of identity of a given Pgroup of a GLP-1 receptor agonist of the invention to SEQ ID NO: 1, 2,and 3, respectively, should happen to be the same, any of those havingthe same highest percentage of identity may be used for thedetermination. An example of such alignment is inserted hereinbelow, inwhich sequence no. 1 is SEQ ID NO: 1, and sequence no. 2 is SEQ ID NO:3:

# 1: GLP_(—)1(9-37) # 2: GLP-1A(3-37) # Matrix: EBLOSUM62 # Gap_penalty:10.0 # Extend_penalty: 0.5 # Length: 35 # Identity: 18/35 (51.4%) #Similarity: 22/35 (62.9%) # Gaps: 6/35 (17.1%) # Score: 92.0

The above alignment is just for illustration, as typically an analogueof one of SEQ ID NOs: 1, 2, or 3 will be compared with either of thesereference sequences.

In case of analogues comprising non-natural amino acids such as Imp,and/or Aib being included in the sequence, these may, for alignmentpurposes, be replaced with X. If desired, X can later be manuallycorrected.

Derivatives

The terms “derivative” as used herein in the context of the GLP-1receptor agonist peptides of the invention means a chemically modifiedpeptide or analogue, in which one or more substituents have beencovalently attached to the peptide. The substituent(s) may also bereferred to as side chain(s). In a particular embodiment, the derivativeof the invention has one side chain. In another particular embodiment ithas two side chains. For the purpose of this definition, the group Y—Z—of formula I is preferably not considered a substituent/side chain.

In particular embodiments, the side chain has at least 10 carbon atoms,or at least 15, 20, 25, 30, 35, 40, or at least 43 carbon atoms. Infurther particular embodiments, the side chain may further include atleast 5 hetero atoms, in particular O and N, for example at least 7, 9,10, 12, 15, 17, or at least 20 hetero atoms, such as at least 1, 2, 3,or 4 N-atoms, and/or at least 3, 4, 6, 9, 12, 13, or 15 O-atoms.

Non-limiting examples of GLP-1 receptor agonist derivatives includeheterologous fusion proteins or conjugates of the GLP-1 receptor agonistpeptides of the invention, with e.g. the Fc portion of an immunoglobulinsuch as IgG, with human albumin, with antibodies such as a glucagonbinding antibody heavy chain variable region, or with fragments oranalogues of any of these (see, e.g., US 2007/0161087, WO 2005/058958,and WO 2007/124463 A2). Additional examples include PEGylated peptides(see, e.g., WO 2005/058954, WO 2004/093823, and WO 2006/124529), as wellas acylated peptides (see, e.g., WO 98/08871, WO2005/027978, WO2006/097537, and WO 2009/030771).

In a preferred embodiment, the side chain is capable of formingnon-covalent aggregates with albumin, thereby promoting the circulationof the derivative with the blood stream, and also having the effect ofprotracting the time of action of the derivative, due to the fact thatthe aggregate of the derivative and albumin is only slowly disintegratedto release the active pharmaceutical ingredient. Thus, a preferredsubstituent, or side chain, as a whole may be referred to as an albuminbinding moiety.

In another particular embodiment, the albumin binding moiety comprises aportion which is particularly relevant for the albumin binding andthereby the protraction, which portion may accordingly be referred to asa protracting moiety. The protracting moiety may be at, or near, theopposite end of the albumin binding moiety, relative to its point ofattachment to the peptide.

In a still further particular embodiment, the albumin binding moietycomprises a portion in-between the protracting moiety and the point ofattachment to the peptide, which portion may be referred to as a linker,linker moiety, spacer, or the like. The presence of a linker isoptional; hence if no linker is present the albumin binding moiety maybe identical to the protracting moiety.

In particular embodiments, the albumin binding moiety and/or theprotracting moiety is lipophilic, and/or negatively charged atphysiological pH (7.4).

The albumin binding moiety, the protracting moiety, or the linker may becovalently attached to a lysine residue of the GLP-1 receptor agonistpeptide by conjugation chemistry such as by alkylation, acylation, esterformation, or amide formation; or to a cysteine residue, such as bymaleimide or haloacetamide (such as bromo-/fluoro-/iodo-) coupling.

In a preferred embodiment, an active ester of the albumin binding moietyand/or the protracting moiety, optionally with a linker, is covalentlylinked to an amino group of a lysine residue, preferably the epsilonamino group thereof, under formation of an amide bond (this processbeing referred to as acylation).

Unless otherwise stated, when reference is made to an acylation of alysine residue, it is understood to be to the epsilon-amino groupthereof.

In one embodiment, the invention relates to a derivative of a GLP-1receptor agonist peptide which comprises, preferably has, an albuminbinding moiety attached to one or more of 18K, 26K, 36K, and/or 37K,wherein reference may be had to the sequence of GLP-1(9-37) (SEQ ID NO:1). As explained above, each residue number refers to the correspondingposition in GLP-1(9-37) (SEQ ID NO: 1). Furthermore, as also explainedabove, ordinary script may be used instead of superscript to designatethe position number. E.g., “K¹⁸” is fully equivalent to “18K”.

Corresponding position numbers are preferably identified by handwritingand eyeballing, or by using a suitable alignment program, as explainedabove.

For the present purposes, the terms “albumin binding moiety”,“protracting moiety”, and “linker” include the un-reacted as well as thereacted forms of these molecules. Whether or not one or the other formis meant is clear from the context in which the term is used.

In one aspect the albumin binding moiety comprises, or consists of, aprotracting moiety selected from

HOOC—(CH₂)_(x)—CO—*  Chem. 8

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 9

R¹⁸—C₆H₄—(CH₂)_(z)—CO—*  Chem. 10

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, and R¹⁸ is a grouphaving a molar mass not higher than 150 Da.

In one embodiment, *—(CH₂)_(x)—* refers to straight or branched,preferably straight, alkylene in which x is an integer in the range of6-18.

In another embodiment, *—(CH₂)_(y)—* refers to straight or branched,preferably straight, alkylene in which y is an integer in the range of3-17.

In a third embodiment, *—(CH₂)_(z)—* refers to straight or branched,preferably straight, alkylene in which z is an integer in the range of1-5.

The molar mass (M) of a chemical substance (such as the group R¹) is themass of one mole of the substance. The molar mass is quoted in dalton,symbol Da, with the definition 1 Da=1 g/mol.

Molar mass may be calculated from standard atomic weights, and is oftenlisted in chemical catalogues. The molar mass of a compound is given bythe sum of the standard atomic weights of the atoms which form thecompound multiplied by the molar mass constant, M_(u) which equals 1g/mol. As an example, the molecular mass of tert. butyl (C₄H₉) isM(C₄H₉)═([4×12.01]+[9×1.008])×1 g/mol=57 Da.

Standard atomic weights are published by the International Union of Pureand Applied Chemistry (IUPAC), and also reprinted in a wide variety oftextbooks, commercial catalogues, wallcharts etc.

For the attachment to the GLP-1 receptor agonist peptide, the acid groupof the fatty acid, or one of the acid groups of the fatty diacid, formsan amide bond with the epsilon amino group of a lysine residue in theGLP-1 receptor agonist peptide.

The term “fatty acid” refers to aliphatic monocarboxylic acids havingfrom 4 to 28 carbon atoms, it is preferably unbranched, and/or evennumbered, and it may be saturated or unsaturated.

The term “fatty diacid” refers to fatty acids as defined above but withan additional carboxylic acid group in the omega position. Thus, fattydiacids are dicarboxylic acids.

The nomenclature is as is usual in the art, for example *—COOH, as wellas HOOC—*, refers to carboxy; *—C₆H₄—* to phenylene; *—CO—*, as well as*—OC—*, to carbonyl (O═C<**); and C₆H₅—O—* to phenoxy.

In particular embodiments, the aromatics, such as the phenoxy and thephenylene radicals, may be, independently, ortho, meta, or para.

In a preferred embodiment the linker moiety, if present, has from 5 to30 C-atoms. In additional preferred embodiments, the linker moiety, ifpresent, has from 4 to 20 hetero atoms. H-atoms are not hetero atoms.

In another embodiment, the linker comprises at least one OEG molecule,at least one glutamic acid residue, and/or at least one piperidinemolecule, optionally substituted, or rather the corresponding radicals(OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. this di-radical:*—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—*).

The amino acid glutamic acid comprises two carboxylic acid groups. Itsgamma-carboxy group is preferably used for forming an amide bond withthe epsilon-amino group of lysine, or with an amino group of an OEGmolecule, if present, or with the amino group of another Glu residue, ifpresent. The amino group of Glu in turn forms an amide bond with thecarboxy group of the protracting moiety, or with the carboxy group of anOEG molecule, if present, or with the gamma-carboxy group of anotherGlu, if present. This way of inclusion of Glu is occasionally brieflyreferred to as “gamma-Glu”.

The derivatives of the invention may exist in different stereoisomericforms having the same molecular formula and sequence of bonded atoms,but differing only in the three-dimensional orientation of their atomsin space. The stereoisomerism of the exemplified derivatives of theinvention is indicated in the experimental section, in the names as wellas the structures, using standard nomenclature. Unless otherwise statedthe invention relates to all stereoisomeric forms of the claimedderivative.

The concentration in plasma of the GLP-1 receptor agonist peptides andderivatives of the invention may be determined using any suitablemethod. For example, LC-MS (Liquid Chromatography Mass Spectroscopy) maybe used, or immunoassays such as RIA (Radio Immuno Assay), ELISA(Enzyme-Linked Immuno Sorbent Assay), and LOCI (Luminescence OxygenChanneling Immunoasssay). General protocols for suitable RIA and ELISAassays are found in, e.g., WO09/030,738 on p. 116-118. A preferred assayis the LOCI assay in which the plasma concentrations of the compoundsare determined using a Luminescence Oxygen Channeling Immunoasssay(LOCI), generally as described for the determination of insulin byPoulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12,p. 240-247. The donor beads are coated with streptavidin, while acceptorbeads are conjugated with a monoclonal antibody recognising amid-/C-terminal epitope of the peptide. Another monoclonal antibody,specific for the N-terminus, is biotinylated. The three reactants arecombined with the analyte and form a two-sited immuno-complex.Illumination of the complex releases singlet oxygen atoms from the donorbeads, which are channeled into the acceptor beads and triggerchemiluminescence which is measured in an Envision plate reader. Theamount of light is proportional to the concentration of the compound.

Pharmaceutically Acceptable Salt, Amide, or Ester

The GLP-1 receptor agonist peptides, derivatives, and intermediateproducts of the invention may be in the form of a pharmaceuticallyacceptable salt, amide, or ester.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.: NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acid salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the peptides and derivatives of the invention may be formedwith added cations or anions that react with anionic or cationic groups,respectively. These groups may be situated in the peptide moiety, and/orin the side chain of the compounds of the invention.

Non-limiting examples of anionic groups of the compounds of theinvention include free carboxylic groups in the side chain, if any, aswell as in the peptide moiety. The peptide moiety often includes a freecarboxylic acid group at the C-terminus, and it may also include freecarboxylic groups at internal acid amino acid residues such as Asp andGlu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

The ester of the peptides and derivatives of the invention may, e.g., beformed by the reaction of a free carboxylic acid group with an alcoholor a phenol, which leads to replacement of at least one hydroxyl groupby an alkoxy or aryloxy group

The ester formation may involve the free carboxylic group at theC-terminus of the peptide, and/or any free carboxylic group in the sidechain.

The amide of the peptides and derivatives of the invention may, e.g., beformed by the reaction of a free carboxylic acid group with an amine ora substituted amine, or by reaction of a free or substituted amino groupwith a carboxylic acid.

The amide formation may involve the free carboxylic group at theC-terminus of the peptide, any free carboxylic group in the side chain,the free amino group at the N-terminus of the peptide, and/or any freeor substituted amino group of the peptide in the peptide and/or the sidechain.

In a particular embodiment, the peptide or derivative is in the form ofa pharmaceutically acceptable salt. In another particular embodiment,the peptide or derivative is in the form of a pharmaceuticallyacceptable amide, preferably with an amide group at the C-terminus ofthe peptide. In a still further particular embodiment, the peptide orderivative is in the form a pharmaceutically acceptable ester.

Intermediate Compounds

The invention also relates to an intermediate product of the formulaChem. 50 or Chem. 51:

wherein Q, R, R1, and R2 are as defined for the GLP-1 receptor agonistpeptide of the invention, having the formula Chem. 1, and each of PG₁and PG₂ represents a protection group.

Non-limiting examples of PG1 groups are Boc, Trt, Mtt, Mmt, and Fmoc.

Non-limiting examples of PG2 groups are —OH, or groups functionalised asan activated ester, for example, without limitation, OPfp, OPnp, andOSuc.

Other suitable activated esters may be selected, e.g., according to theteaching of M. Bodanszky, “Principles of Peptide Synthesis”, 2nd ed.,Springer Verlag, 1993.

Functional Properties

In a first functional aspect, the GLP-1 receptor agonist peptides and/orderivatives of the invention have a good potency. Also, oralternatively, in a second functional aspect, they have a protractedpharmacokinetic profile. Also, or alternatively, in a third functionalaspect, they are stable against degradation by gastro intestinalenzymes. Also, or alternatively, in a fourth functional aspect, theyhave a high oral bioavailability.

Biological Activity (Potency)

According to the first functional aspect, the GLP-1 receptor agonistderivatives are biologically active, or have a good potency.

Surprisingly, they have an improved potency as compared to thecomparative compound of Example 11 (Chem. 40) herein, which is based onone of the most potent compounds of the prior art, viz. compound 215 ofWO2004/067548.

Also, or additionally, the derivatives of the invention have a highbinding affinity to the GLP-1 receptor at low albumin concentration(0.005%), i.e. a low IC₅₀ value, which is discussed further below underthe heading of receptor binding.

As regards potency, the term half maximal effective concentration (EC₅₀)generally refers to the concentration which induces a response halfwaybetween the baseline and maximum, by reference to the dose responsecurve. EC₅₀ is used as a measure of the potency of a compound andrepresents the concentration where 50% of its maximal effect isobserved.

The in vitro potency of the derivatives of the invention may bedetermined as described hereinbelow, and the EC₅₀ of the derivative inquestion determined. The lower the EC₅₀, the better the potency.

In a particular embodiment, the derivatives of the invention are atleast 3 times more potent than Chem. 40; preferably at least 4 timesmore potent; even more preferably at least 5 times more potent; or mostpreferably at least 6 times more potent than Chem. 40.

In another particular embodiment, the derivatives of the invention areat least 7 times more potent than Chem. 40; preferably at least 8 timesmore potent; even more preferably at least 9 times more potent; or mostpreferably at least 10 times more potent than Chem. 40.

In another particular embodiment, the derivatives of the invention areat least 20 times more potent than Chem. 40; preferably at least 50times more potent; even more preferably at least 100 times more potent;still more preferably at least 200 times more potent; or most preferablyat least 400 times more potent than Chem. 40.

Potency is preferably determined as described below, and it is notedthat a, e.g., three times more potent compound has an EC₅₀ which isthree times lower.

In a particular embodiment, potency and/or activity refers to in vitropotency, i.e. performance in a functional GLP-1 receptor assay, more inparticular to the capability of stimulating cAMP formation in a cellline expressing the cloned human GLP-1 receptor.

The stimulation of the formation of cAMP in a medium containing thehuman GLP-1 receptor may preferably be determined using a stabletransfected cell-line such as BHK467-12A (tk-ts13), and/or using for thedetermination of cAMP a functional receptor assay, e.g. based oncompetition between endogenously formed cAMP and exogenously addedbiotin-labelled cAMP, in which assay cAMP is more preferably capturedusing a specific antibody, and/or wherein an even more preferred assayis the AlphaScreen cAMP Assay, most preferably the one described inExample 13.

In an additional particular embodiment, the medium has the followingcomposition (final in-assay concentrations):): 50 mM TRIS-HCl; 5 mMHEPES; 10 mM MgCl₂, 6H₂O; 150 mM NaCl; 0.01% Tween; 0.1% BSA; 0.5 mMIBMX; 1 mM ATP; 1 uM GTP; pH 7.4.

In a further particular embodiment, the GLP-1 receptor agonist hasanEC₅₀ below 2000 pM, preferably below 1800 pM, more preferably below1700 pM, even more preferably below 1600 pM, or most preferably below1500 pM.

In another particular embodiment the derivatives of the invention arepotent in vivo, which may be determined as is known in the art in anysuitable animal model, as well as in clinical trials. The diabetic db/dbmouse is one example of a suitable animal model, and the blood glucoselowering effect may be determined in such mice in vivo, e.g. asdescribed in Example 43 of WO09/030,738.

Protraction—Receptor Binding—High and Low Albumin

According to the second functional aspect, the derivatives of theinvention are protracted.

A suitable assay for determining receptor binding of the peptides andderivatives of the invention at high and low albumin concentration isdisclosed in Example 14 herein. Generally, the binding to the GLP-1receptor at low albumin concentration should be as good as possible,corresponding to a low IC₅₀ value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the compound to the GLP-1receptor. As is known, the peptides GLP-1 receptor agonist peptidederivatives of the invention also bind to albumin. This is a generallydesirable effect, which extends their lifetime in plasma. Therefore, theIC₅₀ value at high albumin will generally be higher than the IC₅₀ valueat low albumin, corresponding to a reduced binding to the GLP-1receptor, caused by albumin binding competing with the binding to theGLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low). On the other hand, albumin bindingmay not always be desirable, or the binding to albumin may become toostrong. Therefore, the desirable ranges for IC₅₀ (low albumin), IC₅₀(high albumin)/, and the ratio high/low may vary from compound tocompound, depending on the intended use and the circumstancessurrounding such use, and on other compound properties of potentialinterest.

In a particular embodiment, the peptides and derivatives of theinvention have a high binding affinity to the GLP-1 receptor at lowalbumin concentration (0.005%), i.e. a low IC₅₀ value.

In a particular embodiment, the GLP-1 receptor binding affinity (IC₅₀)in the presence of 0.005% HSA (low albumin) is below 600.00 nM,preferably below 500.00 nM, more preferably below 200.00 nM, even morepreferably below 100.00 nM, or most preferably below 45.00 nM.

Degradation by Gastro Intestinal Enzymes

According to the third functional aspect, the GLP-1 receptor agonistpeptides and/or derivatives of the invention are stable, or stabilised,against degradation by one or more gastro intestinal enzymes.

Gastro intestinal enzymes include, without limitation, exo and endopeptidases, such as pepsin, trypsin, chymotrypsin, elastases, andcarboxypeptidases. The stability may be tested against these gastrointestinal enzymes in the form of purified enzymes, or in the form ofextracts from the gastrointestinal system.

In a particular embodiment, the derivative of the invention has an invitro half-life (T_(1/2)), in an extract of rat small intestines,divided by the corresponding half-life (T_(1/2)) of GLP-1(7-37), ofabove 1.0, preferably above 2.0, more preferably above 3.0, even morepreferably above 4.0, or most preferably above 5.0. In other words, aratio (SI) may be defined for each derivative, viz. as the in vitrohalf-life (T_(1/2)) of the derivative in question, in an extract of ratsmall intestines, divided by the corresponding half-life (T_(1/2)) ofGLP-1(7-37).

A suitable assay for determining in vitro half-life in an extract of ratsmall intestines is disclosed in Example 57 of a PCT applicationentitled “Double-acylated GLP-1 derivatives” filed with the EPO asreceiving office, by Novo Nordisk A/S, on the same date as the presentapplication.

Protraction—Half Life In Vivo in Rats

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment, protraction may bedetermined as half-life (T_(1/2)) in vivo in rats after i.v.administration. In additional embodiments, the half-life is at least 4hours, preferably at least 5 hours, even more preferably at least 6hours, or most preferably at least 8 hours.

A suitable assay for determining half-life in vivo in rats after i.v.administration is disclosed in Example 58 of a PCT application entitled“Double-acylated GLP-1 derivatives” filed with the EPO as receivingoffice, by Novo Nordisk A/S, on the same date as the presentapplication.

Protraction—Half Life In Vivo in Minipigs

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment protraction may bedetermined as half-life (T_(1/2)) in vivo in minipigs after i.v.administration. In additional embodiments, the half-life is at least 12hours, preferably at least 24 hours, more preferably at least 36 hours,even more preferably at least 48 hours, or most preferably at least 60hours.

A suitable assay for determining half-life in vivo in minipigs afteri.v. administration is disclosed in Example 16 herein.

Oral Bioavailability

According to the fourth functional aspect, the derivatives of theinvention have a high oral bioavailability.

The oral bioavailability of commercial GLP-1 receptor agonist peptidederivatives is very low. The oral bioavailability of such derivativesunder development for i.v. or s.c. administration is also low.

Accordingly, there is a need in the art for derivatives of an improvedoral bioavailability. Such derivatives could be suitable candidates fororal administration, as long as their potency is generally satisfactory,and/or as long as their half-life is also generally satisfactory.

The present inventors identified a novel class of GLP-1 receptor agonistpeptide derivatives, which have a high oral bioavailability, and at thesame time a satisfactory potency, and/or half-life.

Also, or alternatively, these derivatives have a high oralbioavailability, and at the same time a high binding affinity (i.e. alow IC₅₀ value) to the GLP-1 receptor at a low concentration of albumin.

These features are of importance with a view to obtaining a low dailyoral dose of the active pharmaceutical ingredient, which is desirablefor various reasons, including, e.g., economy of production, likelihoodof potential safety issues, as well as administration comfort issues,and environmental concerns.

Generally, the term bioavailability of a GLP-1 receptor agonist compoundof the invention refers to the fraction of an administered dose of thecompound that reaches the systemic circulation unchanged. By definition,when a medication is administered intravenously, its bioavailability is100%. However, when a medication is administered via other routes (suchas orally), its bioavailability decreases (due to incomplete absorptionand first-pass metabolism). Knowledge about bioavailability is essentialwhen calculating dosages for non-intravenous routes of administration.

Absolute oral bioavailability compares the bioavailability (estimated asthe area under the curve, or AUC) of the active drug in systemiccirculation following oral administration, with the bioavailability ofthe same drug following intravenous administration. It is the fractionof the drug absorbed through non-intravenous administration comparedwith the corresponding intravenous administration of the same drug. Thecomparison must be dose normalised if different doses are used;consequently, each AUC is corrected by dividing the corresponding doseadministered.

A plasma drug concentration vs time plot is made after both oral andintravenous administration. The absolute bioavailability (F) is thedose-corrected AUC-oral divided by AUC-intravenous.

The GLP-1 receptor agonist compounds of the invention have an absoluteoral bioavailability which is higher than that of a) liraglutide, and/orb) semaglutide; preferably at least 10% higher, more preferably at least20% higher, even more preferably at least 30% higher, or most preferablyat least 40% higher. Before testing oral bioavailability the GLP-1receptor agonist compounds of the invention may suitably be formulatedas is known in the art of oral formulations of insulinotropic compounds,e.g. using any one or more of the formulations described in WO2008/145728.

A test has been developed, described in Example 15, which was found tobe a very good prediction of oral bioavailability. According to thistest, after direct injection of a GLP-1 derivative into the intestinallumen of rats, the concentration (exposure) thereof in plasma isdetermined, and the ratio of plasma concentration (pmol/l) divided bythe concentration of the dosing solution (umol/l) is calculated for t=30min. This ratio is a measure of intestinal bioavailability, and it hasshown to correlate nicely with actual oral bioavailability data.

Additional particular embodiments of the derivatives of the inventionare described in the section headed “particular embodiments” before theexperimental section.

Production Processes

The production of peptides like for example GLP-1(7-37) and analoguesthereof is well known in the art.

The fragment P of the peptides of formula I of the invention may forinstance be produced by classical peptide synthesis, e.g., solid phasepeptide synthesis using t-Boc or Fmoc chemistry or other wellestablished techniques, see, e.g., Greene and Wuts, “Protective Groupsin Organic Synthesis”, John Wiley & Sons, 1999, Florencio ZaragozaDörwald, “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH,2000, and “Fmoc Solid Phase Peptide Synthesis”, Edited by W. C. Chan andP. D. White, Oxford University Press, 2000.

Also, or alternatively, it may be produced by recombinant methods, viz.by culturing a host cell containing a DNA sequence encoding the fragmentand capable of expressing the peptide in a suitable nutrient mediumunder conditions permitting the expression of the peptide. Non-limitingexamples of host cells suitable for expression of these peptides are:Escherichia coli, Saccharomyces cerevisiae, as well as mammalian BHK orCHO cell lines.

The complete GLP-1 receptor agonist peptides of the inventionincorporating, viz. adding Y—Z to P of formula I, may e.g. be producedas described in the experimental part. Or see Hodgson et al: “Thesynthesis of peptides and proteins containing non-natural amino acids”,Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430; and in WO2009/083549 A1 entitled “Semi-recombinant preparation of GLP-1analogues”.

Derivatives of the invention may be prepared as is known in the art, andspecific examples of methods of preparing a number of derivatives of theinvention are included in the experimental part herein.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a peptide or a derivative of theinvention; or a pharmaceutically acceptable salt, amide, or esterthereof, and a pharmaceutically acceptable excipient may be prepared asis known in the art.

The term “excipient” broadly refers to any component other than theactive therapeutic ingredient(s). The excipient may be an inertsubstance, an inactive substance, and/or a not medicinally activesubstance.

The excipient may serve various purposes, e.g. as a carrier, vehicle,diluent, tablet aid, and/or to improve administration, and/or absorptionof the active substance.

The formulation of pharmaceutically active ingredients with variousexcipients is known in the art, see e.g. Remington: The Science andPractice of Pharmacy (e.g. 19^(th) edition (1995), and any latereditions).

Non-limiting examples of excipients are: Solvents, diluents, buffers,preservatives, tonicity regulating agents, chelating agents, andstabilisers.

Examples of formulations include liquid formulations, i.e. aqueousformulations comprising water. A liquid formulation may be a solution,or a suspension. An aqueous formulation typically comprises at least 50%w/w water, or at least 60%, 70%, 80%, or even at least 90% w/w of water.

Alternatively, a pharmaceutical composition may be a solid formulation,e.g. a freeze-dried or spray-dried composition, which may be used as is,or whereto the physician or the patient adds solvents, and/or diluentsprior to use.

The pH in an aqueous formulation may be anything between pH 3 and pH 10,for example from about 7.0 to about 9.5; or from about 3.0 to about 7.0.

A pharmaceutical composition may comprise a buffer. The buffer may e.g.be selected from the group consisting of sodium acetate, sodiumcarbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine,sodium dihydrogen phosphate, disodium hydrogen phosphate, sodiumphosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malicacid, succinate, maleic acid, fumaric acid, tartaric acid, asparticacid, and mixtures thereof. A pharmaceutical composition may comprise apreservative. The preservative may e.g. be selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol), and mixturesthereof. The preservative may be present in a concentration from 0.1mg/ml to 20 mg/ml. A pharmaceutical composition may comprise an isotonicagent. The isotonic agent may e.g. be selected from the group consistingof a salt (e.g. sodium chloride), a sugar or sugar alcohol, an aminoacid (e.g. glycine, histidine, arginine, lysine, isoleucine, asparticacid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), and mixtures thereof. Any sugar suchas mono-, di-, or polysaccharides, or water-soluble glucans, includingfor example fructose, glucose, mannose, sorbose, xylose, maltose,lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,alfa and beta HPCD, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. Sugar alcohol is defined as aC4-C8 hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one embodiment, the sugar alcohol additive is mannitol.A pharmaceutical composition may comprise a chelating agent. Thechelating agent may e.g. be selected from salts ofethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid,and mixtures thereof. A pharmaceutical composition may comprise astabiliser. The stabiliser may e.g. be one or more oxidation inhibitors,aggregation inhibitors, surfactants, and/or one or more proteaseinhibitors. Non-limiting examples of these various kinds of stabilisersare disclosed in the following.

The term “aggregate formation” refers to a physical interaction betweenthe peptide molecules resulting in formation of oligomers, which mayremain soluble, or large visible aggregates that precipitate from thesolution. Aggregate formation by a peptide during storage of a liquidpharmaceutical composition can adversely affect biological activity ofthat polypeptide, resulting in loss of therapeutic efficacy of thepharmaceutical composition. Furthermore, aggregate formation may causeother problems such as blockage of tubing, membranes, or pumps when thepolypeptide-containing pharmaceutical composition is administered usingan infusion system.

A pharmaceutical composition may comprise an amount of an amino acidbase sufficient to decrease aggregate formation of the polypeptideduring storage of the composition. The term “amino acid base” refers toone or more amino acids (such as methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), oranalogues thereof. Any amino acid may be present either in its free baseform or in its salt form. Any stereoisomer (i.e., L, D, or a mixturethereof) of the amino acid base may be present.

Methionine (or other sulphuric amino acids or amino acid analogous) maybe added to inhibit oxidation of methionine residues to methioninesulfoxide when the polypeptide acting as the therapeutic agent is apolypeptide comprising at least one methionine residue susceptible tosuch oxidation. Any stereoisomer of methionine (L or D) or combinationsthereof can be used.

A the pharmaceutical composition may comprise a stabiliser selected fromthe group of high molecular weight polymers or low molecular compounds.The stabiliser may e.g. be selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). A pharmaceutical composition maycomprise additional stabilising agents such as, but not limited to,methionine and EDTA, which protect the polypeptide against methionineoxidation, and a nonionic surfactant, which protects the polypeptideagainst aggregation associated with freeze-thawing or mechanicalshearing.

A pharmaceutical composition may comprise one or more surfactants,preferably a surfactant, at least one surfactant, or two differentsurfactants. The term “surfactant” refers to any molecules or ions thatare comprised of a water-soluble (hydrophilic) part, and a fat-soluble(lipophilic) part. The surfactant may e.g. be selected from the groupconsisting of anionic surfactants, cationic surfactants, nonionicsurfactants, and/or zwitterionic surfactants.

A pharmaceutical composition may comprise one or more proteaseinhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid),and/or benzamidineHCl.

Additional, optional, ingredients of a pharmaceutical compositioninclude, e.g., wetting agents, emulsifiers, antioxidants, bulkingagents, metal ions, oily vehicles, proteins (e.g., human serum albumin,gelatine), and/or a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine).

Still further, a pharmaceutical composition may be formulated as isknown in the art of oral formulations of insulinotropic compounds, e.g.using any one or more of the formulations described in WO 2008/145728.

An administered dose may contain from 0.01 mg-100 mg of the GLP-1receptor agonist derivative, or from 0.01-50 mg, or from 0.01-20 mg, orfrom 0.01-10 mg of the GLP-1 receptor agonist derivative.

The derivative may be administered in the form of a pharmaceuticalcomposition. It may be administered to a patient in need thereof atseveral sites, for example, at topical sites such as skin or mucosalsites; at sites which bypass absorption such as in an artery, in a vein,or in the heart; and at sites which involve absorption, such as in theskin, under the skin, in a muscle, or in the abdomen.

The route of administration may be, for example, lingual; sublingual;buccal; in the mouth; oral; in the stomach; in the intestine; nasal;pulmonary, such as through the bronchioles, the alveoli, or acombination thereof; parenteral, epidermal; dermal; transdermal;conjunctival; uretal; vaginal; rectal; and/or ocular. A composition maybe an oral composition, and the route of administration is per oral.

A composition may be administered in several dosage forms, for exampleas a solution; a suspension; an emulsion; a microemulsion; multipleemulsions; a foam; a salve; a paste; a plaster; an ointment; a tablet; acoated tablet; a chewing gum; a rinse; a capsule such as hard or softgelatine capsules; a suppositorium; a rectal capsule; drops; a gel; aspray; a powder; an aerosol; an inhalant; eye drops; an ophthalmicointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; avaginal ointment; an injection solution; an in situ transformingsolution such as in situ gelling, setting, precipitating, and in situcrystallisation; an infusion solution; or as an implant. A compositionmay be a tablet, optionally coated, a capsule, or a chewing gum.

A composition may further be compounded in a drug carrier or drugdelivery system, e.g. in order to improve stability, bioavailability,and/or solubility. In a particular embodiment a composition may beattached to such system through covalent, hydrophobic, and/orelectrostatic interactions. The purpose of such compounding may be,e.g., to decrease adverse effects, achieve chronotherapy, and/orincrease patient compliance.

A composition may also be used in the formulation of controlled,sustained, protracting, retarded, and/or slow release drug deliverysystems.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal, or intravenous injection by means of asyringe, optionally a pen-like syringe, or by means of an infusion pump.

A composition may be administered nasally in the form of a solution, asuspension, or a powder; or it may be administered pulmonally in theform of a liquid or powder spray.

Transdermal administration is a still further option, e.g. byneedle-free injection, from a patch such as an iontophoretic patch, orvia a transmucosal route, e.g. buccally.

A composition may be a stabilised formulation. The term “stabilisedformulation” refers to a formulation with increased physical and/orchemical stability, preferably both. In general, a formulation must bestable during use and storage (in compliance with recommended use andstorage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

The treatment with a derivative according to the present invention mayalso be combined with one or more additional pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. Examples of these pharmacologically active substances are:Insulin, sulphonylureas, biguanides, meglitinides, glucosidaseinhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)inhibitors, inhibitors of hepatic enzymes involved in stimulation ofgluconeogenesis and/or glycogenolysis, glucose uptake modulators,compounds modifying the lipid metabolism such as antihyperlipidemicagents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides(GIP analogs), compounds lowering food intake, RXR agonists and agentsacting on the ATP-dependent potassium channel of the β-cells;Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide,repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, alatriopril, quinapril and ramipril, calcium channelblockers such as nifedipine, felodipine, nicardipine, isradipine,nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin,urapidil, prazosin and terazosin; CART (cocaine amphetamine regulatedtranscript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, Y2receptor agonists, Y4 receptor agonits, mixed Y2/Y4 receptor agonists,MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosisfactor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP(corticotropin releasing factor binding protein) antagonists, urocortinagonists, agonists, oxyntomodulin and analogues, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists, Gastric Inhibitory Polypeptide agonists or antagonists (GIPanalogs), gastrin and gastrin analogs.

The treatment with a derivative according to this invention may also becombined with a surgery that influences the glucose levels, and/or lipidhomeostasis such as gastric banding or gastric bypass.

Pharmaceutical Indications

The present invention also relates to a GLP-1 receptor agonist peptideof the invention, and a derivative thereof, for use as a medicament.

In particular embodiments, these compounds may be used for the followingmedical treatments, all preferably relating one way or the other todiabetes:

(i) prevention and/or treatment of all forms of diabetes, such ashyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetesof the young), gestational diabetes, and/or for reduction of HbA1C;

(ii) delaying or preventing diabetic disease progression, such asprogression in type 2 diabetes, delaying the progression of impairedglucose tolerance (IGT) to insulin requiring type 2 diabetes, and/ordelaying the progression of non-insulin requiring type 2 diabetes toinsulin requiring type 2 diabetes;

(iii) improving β-cell function, such as decreasing β-cell apoptosis,increasing β-cell function and/or β-cell mass, and/or for restoringglucose sensitivity to β-cells; (iv) prevention and/or treatment ofcognitive disorders;

(v) prevention and/or treatment of eating disorders, such as obesity,e.g. by decreasing food intake, reducing body weight, suppressingappetite, inducing satiety; treating or preventing binge eatingdisorder, bulimia nervosa, and/or obesity induced by administration ofan antipsychotic or a steroid; reduction of gastric motility; and/ordelaying gastric emptying;

(vi) prevention and/or treatment of diabetic complications, such asneuropathy, including peripheral neuropathy; nephropathy; orretinopathy;

(vii) improving lipid parameters, such as prevention and/or treatment ofdyslipidemia, lowering total serum lipids; lowering HDL; lowering small,dense LDL; lowering VLDL: lowering triglycerides; lowering cholesterol;increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in ahuman; inhibiting generation of apolipoprotein a (apo(a)) in vitroand/or in vivo;

(iix) prevention and/or treatment of cardiovascular diseases, such assyndrome X; atherosclerosis; myocardial infarction; coronary heartdisease; stroke, cerebral ischemia; an early cardiac or earlycardiovascular disease, such as left ventricular hypertrophy; coronaryartery disease; essential hypertension; acute hypertensive emergency;cardiomyopathy; heart insufficiency; exercise tolerance; chronic heartfailure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis;mild chronic heart failure; angina pectoris; cardiac bypass reocclusion;intermittent claudication (atheroschlerosis oblitterens); diastolicdysfunction; and/or systolic dysfunction;

(ix) prevention and/or treatment of gastrointestinal diseases, such asinflammatory bowel syndrome; small bowel syndrome, or Crohn's disease;dyspepsia; and/or gastric ulcers;

(x) prevention and/or treatment of critical illness, such as treatmentof a critically ill patient, a critical illness poly-nephropathy (CIPNP)patient, and/or a potential CIPNP patient; prevention of criticalillness or development of CIPNP; prevention, treatment and/or cure ofsystemic inflammatory response syndrome (SIRS) in a patient; and/or forthe prevention or reduction of the likelihood of a patient sufferingfrom bacteraemia, septicaemia, and/or septic shock duringhospitalisation; and/or

(xi) prevention and/or treatment of polycystic ovary syndrome (PCOS).

In a particular embodiment, the indication is selected from the groupconsisting of (i)-(iii) and (v)-(iix), such as indications (i), (ii),and/or (iii); or indication (v), indication (vi), indication (vii),and/or indication (iix).

In another particular embodiment, the indication is (i). In a furtherparticular embodiment the indication is (v). In a still furtherparticular embodiment the indication is (iix).

The following indications are particularly preferred: Type 2 diabetes,and/or obesity.

PARTICULAR EMBODIMENTS

The following are particular embodiments of the invention:

1. A GLP-1 receptor agonist peptide having the formula Chem. 1:

Y—Z—P,  Chem. 1

whereinP represents a fragment of a GLP-1 receptor agonist peptide lacking thetwo N-terminal amino acid residues;Z represents a group of the formula Chem. 2:

-   -   wherein    -   W represents a group of formula Chem. 3:

-   -   -   wherein            -   R1 and R2 independently represent                -   (i) hydrogen, alkyl, aryl, heterocyclyl, heteroaryl,                    halogen, hydroxyl, hydroxylalkyl, cyano, amino,                    aminoalkyl, carboxyl, carboxylalkyl, alkoxy,                    aryloxy, carboxamide, substituted carboxamide, alkyl                    ester, aryl ester, alkyl sulfonyl, or aryl sulfonyl,                    or            -   R1 and R2 together form                -   (ii) cyclo alkyl, heterocyclyl, or heteroaryl, with                    the proviso that                -   (iii) R1 and R2 do not both represent hydrogen;                    and                    Y represents a group of formula Chem. 4 or Chem. 5:

wherein

-   -   X₁ is N, O, or S; X₂, X₃, X₄, and X₅ independently represent C,        or N, with the proviso that at least one of X₂, X₃, X₄ and X₅ is        C;    -   R11, R12, R13, and R14 independently represent        -   hydrogen, alkyl, aryl, heterocyclyl, heteroaryl, halogen,            hydroxyl, hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl,            carboxylalkyl, alkoxy, aryloxy, carboxamide, substituted            carboxamide, alkyl ester, aryl ester, alkyl sulfonyl, or            aryl sulfonyl;    -   Q represents a bond, or a group of formula Chem. 6:

*—(C(R15)(R16))_(q)—*,  Chem. 6

-   -   -   -   wherein                -   q is 1-6, and                -   R15 and R16 independently of each other and                    independently for each value of q represent                    hydrogen, alkyl, carboxyl, or hydroxyl; and

    -   R represents hydrogen, or alkyl;        or a pharmaceutically acceptable salt, amide, or ester thereof.        2. The peptide of embodiment 1, wherein R1 and R2 independently        represent hydrogen, alkyl, aryl, halogen, hydroxyl,        hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl,        alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester;        wherein preferably alkyl, hydroxylalkyl, aminoalkyl,        carboxylalkyl, alkoxy, and/or alkyl ester contains lower alkyl,        straight or branched, more preferably having 1-6 C-atoms.        3. The peptide of any one of embodiments 1-2, wherein R1 and R2        independently represent hydrogen, lower alkyl, or lower alkoxy,        wherein the lower alkyl and lower alkoxy, independently, have        1-5 C-atoms, preferably 1-4 C-atoms, or most preferably 1-3        C-atoms.        4. The peptide of any one of embodiments 1-3, wherein R1 and R2        independently represent hydrogen, alkyl having 1-2 C-atoms        (ethyl, or methyl), or alkoxy having 1-2 C-atoms (ethoxy,        methoxy).        5. The peptide of any one of embodiments 1-4, wherein R1 and R2        are methyl or methoxy, preferably methyl.        6. The peptide of embodiment 1, wherein R1 and R2 independently        represent alkyl, aryl, halogen, hydroxyl, hydroxylalkyl, amino,        aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,        carboxamide, alkyl ester, or aryl ester; wherein preferably        alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy, and/or        alkyl ester contains lower alkyl, straight or branched, more        preferably having 1-6 C-atoms.        7. The peptide of any one of embodiments 1 and 6, wherein R1 and        R2 independently represent lower alkyl, or lower alkoxy, wherein        the lower alkyl and lower alkoxy, independently, have 1-5        C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms.        8. The peptide of any one of embodiments 1, and 6-7, wherein R1        and R2 independently represent alkyl having 1-2 C-atoms (ethyl,        or methyl), or alkoxy having 1-2 C-atoms (ethoxy, methoxy).        9. The peptide of any one of embodiments 1, and 6-8, wherein R1        and R2 are methyl or methoxy, preferably methyl.        10. The peptide of any one of embodiments 1-9, wherein Z is a        group of formula Chem. 7):

11. The peptide of any one of embodiments 1-10, wherein Y is Chem. 4.12. The peptide of any one of embodiments 1-11, wherein X₁ is N.13. The peptide of any one of embodiments 1-12, wherein one of X₂, X₃,X₄, and X₅ is N.14. The peptide of any one of embodiments 1-13, preferably the peptideof embodiment 12, wherein i) X₃ is N; ii) X₄ is N; iii) X₅ is N; or X₂is N.15. The peptide of embodiment any one of embodiments 1-14, wherein R11and R12 independently represent hydrogen, alkyl, aryl, halogen,hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl,alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester; whereinpreferably alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy,and/or alkyl ester contains lower alkyl, straight or branched, morepreferably having 1-6 C-atoms.16. The peptide of any one of embodiment 1-15, wherein R11 and R12independently represent hydrogen, lower alkyl, or lower alkoxy, whereinthe lower alkyl and lower alkoxy, independently, have 1-5 C-atoms,preferably 1-4 C-atoms, or most preferably 1-3 C-atoms.17. The peptide of any one of embodiments 1-16, wherein R11 and R12independently represent hydrogen, alkyl having 1-2 C-atoms (ethyl, ormethyl), or alkoxy having 1-2 C-atoms (ethoxy, methoxy).18. The peptide of any one of embodiments 1-17, wherein R11 and R12 aremethyl or hydrogen, preferably hydrogen.19. The peptide of any one of embodiments 1-18, wherein Y is aderivative of 1H-imidazole, preferably 1H-imidazol-4-yl, optionallysubstituted at one or two of positions 2, 3, and/or 5, wherein theposition numbering of imidazole is according to IUPAC, and/or as shownfor imidazole on wikipedia on 3 Dec. 2010 at 18:00 DK time.20. The peptide of any one of embodiments 1-19, wherein Q is attached toi) X₂, ii) X₃, X₄, or X₅, preferably to X₂ or X₄, most preferably to X₄.21. The peptide of any one of embodiments 1-20, wherein q is 1-5,preferably 1-4, more preferably 1-3, even more preferably 1, 2, or 3.22. The peptide of any one of embodiments 1-21, wherein R15 and R16independently of each other and independently for each value of qrepresent hydrogen.23. The peptide of any one of embodiments 1-22, wherein R15 and R16 bothrepresent hydrogen.24. The peptide of any one of embodiments 1-23, wherein R is hydrogen.25. The peptide of any one of embodiments 1-24, wherein Y is aderivative of an imidazole, such as 1H-imidazol, being substituted witha group of formula Chem. 7:

*—(R17)—N—*,  Chem. 7

wherein R17 represents alkylene, straight or branched, having 1-6C-atoms, preferably 1-5 C-atoms, more preferably 1-4 C-atoms, or mostpreferably 1-3 C-atoms.26. The peptide of any one of embodiments 1-10, wherein Y is Chem. 5.

27. The peptide of embodiment any one of embodiments 1-10 and 26,wherein R13 and R14 independently represent hydrogen, alkyl, aryl,halogen, hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl,carboxylalkyl, alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester;wherein preferably alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl,alkoxy, and/or alkyl ester contains lower alkyl, straight or branched,more preferably having 1-6 C-atoms.

28. The peptide of any one of embodiment 1-10 and 26-27, wherein R13 andR14 independently represent hydrogen, lower alkyl, or lower alkoxy,wherein the lower alkyl and lower alkoxy, independently, have 1-5C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms.29. The peptide of any one of embodiments 1-10 and 26-28, wherein R13and R14 independently represent hydrogen, alkyl having 1-2 C-atoms(ethyl, or methyl), or alkoxy having 1-2 C-atoms (ethoxy, methoxy).30. The peptide of any one of embodiments 1-10 and 26-29, wherein R13and R14 are methyl or hydrogen, preferably hydrogen.31. The peptide of any one of embodiments 1-10 and 26-30, wherein Y is aderivative of pyridine, preferably pyridin-2-yl, optionally substitutedat one or two of positions 3, 4, 5, and/or 6, where the positionnumbering of pyridine is according to IUPAC, and/or as shown forpyridine on wikipedia on 3 Dec. 2010 at 18:00 DK time.32. The peptide of any one of embodiments 1-10 and 26-31, wherein Q isattached to position 2, 3, 4, 5, or 6 of the pyridine ring, preferablyto position 2, where the position numbering of pyridine is as defined inembodiment 31.33. The peptide of any one of embodiments 1-32, wherein q is as definedin embodiment 21.34. The peptide of any one of embodiments 1-33, wherein R15 and R16 areas defined in any one of embodiments 22-23.35. The peptide of any one of embodiments 1-34, wherein R is hydrogen.36. The peptide of any one of embodiments 1-35, wherein the GLP-1receptor agonist peptide of which P is a fragment lacking the twoN-terminal amino acid residues is an agonist of the human GLP-1receptor, wherein agonist activity is preferably determined asstimulation of the formation of cAMP in a medium containing the humanGLP-1 receptor.37. The peptide of embodiment 36, wherein the medium is a suitablemedium, such as a medium containing the human GLP-1 receptor and havingthe following composition (final in-assay concentrations): 50 mMTris-HCl, 1 mM EGTA, 1.5 mM MgSO₄, 1.7 mM ATP, 20 mM GTP, 2 mM3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, pH 7.4; morepreferably the following composition (final in-assay concentrations): 50mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl₂, 6H₂O; 150 mM NaCl; 0.01% Tween;0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; pH 7.4.38. The peptide of any one of embodiments 1-37, wherein the GLP-1receptor agonist peptide of which P is a fragment lacking the twoN-terminal amino acid residues has a potency corresponding to an EC₅₀ ofbelow 1000 pM, preferably below 500 pM, more preferably below 250 pM,even more preferably below 125 pM, or most preferably below 50 pM.39. The peptide of any one of embodiments 1-38, wherein the GLP-1receptor agonist peptide of which P is a fragment lacking the twoN-terminal amino acid residues is selected from His-Ala-“P”,His-Gly-“P”, and “His-Ser-P”.40. The peptide of any one of embodiments 1-39, wherein the GLP-1receptor agonist peptide of which P is a fragment lacking the twoN-terminal amino acid residues is selected from His-Ala-“P” andHis-Gly-“P”.41. The peptide of any one of embodiments 1-40, wherein P is selectedfrom i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2),iii) GLP-1A(3-37) (SEQ ID NO: 3), and iv) analogues of i), ii), or iii)having a maximum of eight amino acid residues exchanged as compared tothe respective sequence i), ii), or iii) with which the analogue has thehighest similarity, or, preferably, percentage of identity.42. The peptide of any one of embodiments 1-41, wherein P is GLP-1(9-37)(SEQ ID NO: 1), or an analogue thereof having a maximum of eight aminoacid residues exchanged as compared to SEQ ID NO: 1.43. The peptide of any one of embodiments 1-42, wherein P has a) amaximum of seven, six, or five; preferably b) a maximum of four, or c)most preferably a maximum of three amino acid residues exchanged, ascompared to one of the sequences of i) GLP-1(9-37) (SEQ ID NO: 1), ii)exendin-4(3-39) (SEQ ID NO: 2), and iii) GLP-1A(3-37) (SEQ ID NO: 3),with which P has the highest similarity, wherein preferably thecomparison is made with GLP-1(9-37) (SEQ ID NO: 1), or withGLP-1A(3-37), most preferably with GLP-1(9-37).44. The peptide of any one of embodiments 1-43, wherein P has a maximumof two, preferably a maximum of one, or most preferably no amino acidresidues exchanged as compared to one of the sequences of i) GLP-1(9-37)(SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii)GLP-1A(3-37) (SEQ ID NO: 3), with which P has the highest similarity,wherein preferably the comparison is made with GLP-1(9-37) (SEQ ID NO:1), or with GLP-1A(3-37), most preferably with GLP-1(9-37).45. The peptide of any one of embodiments 1-44, which has a C-terminalamide.46. The peptide of any one of embodiments 1-45, which has a C-terminal—COOH group.47. The peptide of any one of embodiments 1-46, which comprises at leastone of the following substitutions as compared to GLP-1(9-37) (SEQ IDNO: 1): 18K; 22E; 30E; 31H; 34Q,R; 36K; 37K; and/or 38E.48. The peptide of any one of embodiments 1-47, which comprises 18K.49. The peptide of any one of embodiments 1-48, which comprises 22E.50. The peptide of any one of embodiments 1-49, which comprises 30E.51. The peptide of any one of embodiments 1-50, which comprises 31H.52. The peptide of any one of embodiments 1-51, which comprises 34Q or34R, preferably 34R.53. The peptide of any one of embodiments 1-52, which comprises 36K.54. The peptide of any one of embodiments 1-53, which comprises 37K.55. The peptide of any one of embodiments 1-54, which comprises 38E.56. The peptide of any one of embodiments 1-55, which comprises 34R and37K.57. The peptide of any one of embodiments 1-56, which comprises 30E and36K.58. The peptide of any one of embodiments 1-57, which comprises 31H and34Q.59. The peptide of any one of embodiments 56-57, which further comprises38E.60. The peptide of any one of embodiments 1-59 which comprises thefollowing substitutions: (i) 18K, 22E, 34Q; (ii) 31H, 34Q; (iii) 30E,36K; (iv) 30E, 36K, 38E; (v) 34R; (vi) 34R, 37K; or (vii) 34R, 37K, 38E.61. The peptide of any one of embodiments 1-60, which has the followingsubstitutions as compared to GLP-1(9-37) (SEQ ID NO: 1), all other aminoacid residues being as in SEQ ID NO: 1: (i) 18K, 22E, 34Q; (ii) 31H,34Q; (iii) 30E, 36K; (iv) 30E, 36K, 38E; (v) 34R; (vi) 34R, 37K; of(vii) 34R, 37K, 38E.62. The peptide of any one of embodiments 1-46, which comprises at leastone of the following substitutions as compared to GLP-1A(3-37) (SEQ IDNO: 3): 17Q,R; 20R; 33R; and/or 38K.63. The peptide of any one of embodiments 1-46, and 62, which has thefollowing substitutions, as compared to GLP-1A(3-37) (SEQ ID NO: 3), allother amino acid residues being as in SEQ ID NO: 3: 17R, 20R, 33R, and38K.64. A GLP-1 receptor agonist peptide selected from the following:

-   (i)    N⁹-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-Lys¹⁸,Glu²²,Gln³⁴]GLP-1(9-37)-peptide;-   (ii)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[His³¹,Gln³⁴]GLP-1    (9-37)-peptide;-   (iii)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)-Glu³⁸-peptide    amide;-   (iv)    N⁹-{2-[2-(1H-Imidazol-4-yl)-propylcarbamoyl]-2-methyl-propionyl}-Glu³⁰,Lys³⁶]GLP-1    (9-37)Glu³⁸-peptide amide;-   (v)    N⁹-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)-Glu³⁸-peptide    amide;-   (vi)    N⁹-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Arg¹⁷,Arg²⁰,Arg³³,Lys³⁸]GLP-1A(3-37)-peptide;-   (vii)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)-peptide;-   (iix)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg³⁴,    Lys³⁷]GLP-1 (9-37)Glu³⁸-peptide;-   (ix)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg³⁴,    Lys³⁷]GLP-1 (9-37)-peptide;-   (x)    N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg³]GLP-1(9-37)-peptide;    and-   (xii)    N⁹-[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl][Arg³⁴,Lys³⁷]-GLP-1-(9-37)-peptide;    or a pharmaceutically acceptable salt, amide, or ester thereof.    64. The peptide of any one of embodiments 1-63 which has a maximum    of two K residues.    65. The peptide of any one of embodiments 1-64, which has a maximum    of one K residue.    66. The peptide of any one of embodiments 1-65, wherein

a) the position corresponding to any of the indicated positions of i)GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii)GLP-1A(3-37) (SEQ ID NO: 3); and/or

b) the number of amino acid modifications as compared to i) GLP-1(9-37)(SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii)GLP-1A(3-37) (SEQ ID NO: 3), is/are identified by handwriting andeyeballing.

67. The peptide of any one of embodiments 1-66, wherein

a) the position corresponding to any of the indicated positions of i)GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii)GLP-1A(3-37) (SEQ ID NO: 3); and/or

b) the number of amino acid modifications as compared to i) GLP-1(9-37)(SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ ID NO: 2), and iii)GLP-1A(3-37) (SEQ ID NO: 3), is/are identified by use of a standardprotein or peptide alignment program.

68. The peptide of embodiment 67, wherein the alignment program is aNeedleman-Wunsch alignment.69. The peptide of any one of embodiments 67-68, wherein the defaultscoring matrix and the default identity matrix is used.70. The peptide of any one of embodiments 67-69, wherein the scoringmatrix is BLOSUM62.71. The peptide of any one of embodiments 67-70, wherein the penalty forthe first residue in a gap is −10 (minus ten).72. The peptide of any one of embodiments 67-71, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).73. The peptide of any one of embodiments 1-72, wherein, a residuenumber, preferably any residue number, be it in superscript after anamino acid residue, or in ordinary script before or after the amino acidresidue in question, refers to the corresponding position in one of thesequences of i) GLP-1(9-37) (SEQ ID NO: 1), ii) exendin-4(3-39) (SEQ IDNO: 2), and iii) GLP-1A(3-37) (SEQ ID NO: 3).74. A derivative of a peptide of any one of embodiments 1-73, or apharmaceutically acceptable salt, amide, or ester thereof.75. The derivative of embodiment 74 which comprises, preferably has, analbumin binding moiety attached to a lysine residue, more preferably tothe epsilon-amino group thereof, via an amide bond.76. The derivative of embodiment 75, which comprises, preferably has, analbumin binding moiety attached to one or more of 18K, 26K, 36K, and/or37K; preferably one albumin binding moiety attached to 18K, 26K, or 36K;or two albumin binding moieties attached to 26K and 37K; whereinreference may be had to the sequence of GLP-1(9-37) (SEQ ID NO: 1).77. The derivative of embodiment 75, which has an albumin binding moietyattached to one or more of 17K, 20K, 33K, and/or 38K; preferably onealbumin binding moiety attached to 38K; or two albumin binding moietiesattached to 20K and 33K; wherein reference may be had to the sequence ofGLP-1A(3-37) (SEQ ID NO: 3).78. The derivative of any one of embodiments 75-77, in which the albuminbinding moiety comprises a protracting moiety.79. The derivative of embodiment 78, wherein

the protracting moiety is selected from Chem. 8, Chem. 9, and Chem. 10:

HOOC—(CH₂)_(x)—CO—*  Chem. 8

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 9

R¹⁸—C₆H₄—(CH₂)_(z)—CO—*  Chem. 10

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17, z is an integer in the range of 1-5, and R¹⁸ is a grouphaving a molar mass not higher than 150 Da.

80. The derivative of embodiment 79, in which the protracting moiety isChem. 8, in which x is an even number, preferably in the range of 14-18,more preferably 16-18, or most preferably x is 16.81. The derivative of embodiment 79, in which the protracting moiety isChem. 9, in which y is an odd number, preferably in the range of 7-15.82. The derivative of embodiment 79, in which the protracting moiety isChem. 10, in which z is an odd number, preferably 3; and R¹⁸ is tert.butyl.83. The derivative of any one of embodiments 79 and 81, in which y is 9.84. The derivative of any one of embodiments 79, 81, and 83, in whichthe —COOH group is in the para-position.85. The derivative of any one of embodiments 79, and 82, in which R¹⁸ isin the para-position.86. The derivative of any one of embodiments 75-84 which comprises alinker.87. The derivative of any one of embodiments 75-86, wherein the albuminbinding moiety comprises a linker.88. The derivative of any one of embodiments 75-87, wherein the albuminbinding moiety further comprises a linker.89. The derivative of any one of embodiments 86-88, wherein the linkeris a di-radical which comprises an N radical and a CO radical, whereini) the N-radical is represented by a first *—NR¹⁹R²⁰ group, where R¹⁹and R²⁰ may, independently, designate hydrogen, carbon, or sulphur,optionally substituted; andii) the CO radical is represented by a first *—CO group,and wherein, preferably, the first *—NR¹⁹R²⁰ group is capable of formingan amide bond with a second *—CO group, and the first *—CO group iscapable of forming an amide bond with a second *—NR¹⁹R²⁰ group, whereinthe second *—NR¹⁹R²⁰ group and the second *—CO group are defined as thefirst *—NR¹⁹R²⁰ group and the first *—CO group, respectively, and formpart, independently, of the structure of i) the analogue, ii) theprotracting moiety, and/or iii) another linker.90. The derivative of any one of embodiments 74-89, which comprises atleast one linker selected from the group consisting of Chem. 11, Chem.12, Chem. 13, and Chem. 14:

*—NH—CH₂—CH₂—(O—CH₂—CH₂)_(k)—O—(CH₂)_(n)—CO—*  Chem. 11

*—NH—C(COOH)—(CH₂)₂—CO—*  Chem. 12

*—N—C((CH₂)₂COOH)—CO—*  Chem. 13

*—NC₅H₈—CO—*  Chem. 14

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; and wherein Chem. 12 and Chem. 13 are di-radicals of Glu.91. The derivative of embodiment 90, wherein the linker comprises Chem.11, and wherein preferably Chem. 11 is a first linker element.92. The derivative of any one of embodiments 90-91, wherein k is 1.93. The derivative of any one of embodiments 90-92, wherein n is 1.94. The derivative of any one of embodiments 90-93, wherein Chem. 11 isincluded m times, wherein m is an integer in the range of 1-10.95. The derivative of embodiment 94, wherein m is an integer in therange of 1-6; preferably in the range of 1-4; more preferably m is 1 or2; even more preferably m is 1; or most preferably m is 2.96. The derivative of any one of embodiments 94-95, wherein, when m isdifferent from 1, the Chem. 11 elements are interconnected via amidebond(s).97. The derivative of any one of embodiments 90-96, wherein the linkerconsists of one or more Chem. 11 elements.98. The derivative of any one of embodiments 90-97, wherein Chem. 11 isrepresented by Chem. 11a:

wherein k and n are as defined in any one of embodiments 90-97.99. The derivative of any one of embodiments 90-99, wherein the linkercomprises a Glu di-radical, such as Chem. 12, and Chem. 13.100. The derivative of any one of embodiments 90-99, wherein Chem. 12and Chem. 13, independently, may be represented by Chem. 12a and Chem.13a, respectively:

most preferably by Chem. 12a.101. The derivative of any one of embodiments 90-101, wherein the Gludi-radical, such as Chem. 12, and/or Chem. 13, independently, isincluded p times, wherein p is an integer in the range of 1-3.102. The derivative of embodiment 101, wherein p is 1, 2, or 3;preferably 1 or 2, or most preferably 1.103. The derivative of any one of embodiments 90-102, wherein the Gludi-radical is a radical of L-Glu or D-Glu, preferably of L-Glu.104. The derivative of any one of embodiments 90-103, wherein the linkerconsists of a Glu di-radical, preferably Chem. 12, more preferably Chem.12a.105. The derivative of any one of embodiments 90-104, wherein the linkercomprises Chem. 14.106. The derivative of any one of embodiments 90-105, where Chem. 14 isrepresented by Chem. 14a:

107. The derivative of any one of embodiments 90-106, wherein the linkerconsists of Chem. 11, being connected at its *—NH end to * the *—CO endof the protracting moiety, and at its *—CO end to the epsilon aminogroup of a lysine residue of the peptide.108. The derivative of any one of embodiments 90-106, wherein the linkerconsists of one time Chem. 12 and two times Chem. 11, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of a lysine residue of the peptide.109. The derivative of any one of embodiments 90-106, wherein the linkerconsists of one time Chem. 14, one time Chem. 12, and two times Chem.11, interconnected via amide bonds and in the sequence indicated, thelinker being connected at its *—NH end to the *—CO end of theprotracting moiety, and at its *—CO end to the epsilon amino group of alysine residue of the peptide.110. The derivative of any one of embodiments 90-109, wherein the one ormore linker(s) are interconnected via amide bond(s).111. A compound selected from the following: Chem. 30, Chem. 31, Chem.32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem.39, and Chem. 41; or a pharmaceutically acceptable salt, amide, or esterthereof.112. A compound characterised by its name, and selected from a listingof each of the names of the compounds of Examples 1-10, and 12 herein;or a pharmaceutically acceptable salt, amide, or ester thereof.113. The compound of embodiment 112, which is a compound of embodiment111.114. The compound of any one of embodiments 111-113, which is aderivative of any one of embodiments 74-110.115. The peptide or derivative of any one of embodiments 1-114, whichhas GLP-1 activity.116. The peptide or derivative of embodiment 115, wherein GLP-1 activityrefers to the capability of activating the human GLP-1 receptor.117. The peptide or derivative of embodiment 116, wherein activation ofthe human GLP-1 receptor is measured in an in vitro assay, as thepotency of cAMP production.118. The peptide or derivative of any one of embodiments 1-117, whichhas a potency corresponding to an EC₅₀ at or below 4500 pM, preferablybelow 4500 pM, more preferably below 4000 pM, even more preferably below3500 pM, or most preferably below 3000 pM.119. The peptide or derivative of any one of embodiments 1-118, whichhas a potency corresponding to an EC₅₀ below 2500 pM, preferably below2000 pM, more preferably below 1500 pM, even more preferably below 1000pM, or most preferably below 800 pM.120. The peptide or derivative of any one of embodiments 1-119 which hasa potency corresponding to an EC₅₀ below 600 pM, preferably below 500pM, more preferably below 400 pM, even more preferably below 300 pM, ormost preferably below 200 pM.121. The peptide or derivative of any one of embodiments 1-120 which hasa potency corresponding to an EC₅₀ below 180 pM, preferably below 160pM, more preferably below 140 pM, even more preferably below 120 pM, ormost preferably below 100 pM.122. The peptide or derivative of any one of embodiments 1-121 which hasa potency corresponding to an EC₅₀ below 80 pM, preferably below 60 pM,more preferably below 50 pM, even more preferably below 40 pM, or mostpreferably below 30 pM.123. The peptide or derivative of any one of embodiments 1-122, whereinthe potency is determined as EC₅₀ for the dose-response curve showingdose-dependent formation of cAMP in a medium containing the human GLP-1receptor, preferably using a stable transfected cell-line such asBHK467-12A (tk-ts13), and/or using for the determination of cAMP afunctional receptor assay, e.g. based on competition betweenendogenously formed cAMP and exogenously added biotin-labelled cAMP, inwhich assay cAMP is more preferably captured using a specific antibody,and/or wherein an even more preferred assay is the AlphaScreen cAMPAssay, most preferably the one described in Example 13.124. The peptide or derivative of any one of embodiments 1-123, the EC₅₀of which is less than 10 times the EC₅₀ of semaglutide, preferably lessthan 8 times the EC₅₀ of semaglutide, more preferably less than 6 timesthe EC₅₀ of semaglutide, even more preferably less than 4 times the EC₅₀of semaglutide, or most preferably less than 2 times the EC₅₀ ofsemaglutide.125. The peptide or derivative of any one of embodiments 1-124, the EC₅₀of which is less than the EC₅₀ of semaglutide, preferably less than 0.8times the EC₅₀ of semaglutide, more preferably less than 0.6 times thepotency of semaglutide, even more preferably less than 0.4 times theEC₅₀ of semaglutide, or most preferably less than 0.2 times the EC₅₀ ofsemaglutide.126. The peptide or derivative of any one of embodiments 1-125, the EC₅₀of which is less than 10 times the EC₅₀ of liraglutide, preferably lessthan 8 times the EC₅₀ of liraglutide, more preferably less than 6 timesthe EC₅₀ of liraglutide, even more preferably less than 4 times the EC₅₀of liraglutide, or most preferably less than 2 times the EC₅₀ ofliraglutide.127. The peptide or derivative of any one of embodiments 1-126, the EC₅₀of which is less than the EC₅₀ of liraglutide, preferably less than 0.8times the EC₅₀ of liraglutide, more preferably less than 0.6 times theEC₅₀ of liraglutide, even more preferably less than 0.5 times the EC₅₀of liraglutide, or most preferably less than or at 0.4 times the EC₅₀ ofliraglutide.128. The derivative of any one of embodiments 74-127, for which theratio [GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0%HSA (high albumin), divided by GLP-1 receptor binding affinity (IC₅₀) inthe presence of 0.005% HSA (low albumin)] is:a) at least 0.5, preferably at least 1.0, more preferably at least 10,even more preferably at least 20, or most preferably at least 30;b) at least 40, preferably at least 50, more preferably at least 60,even more preferably at least 70, or most preferably at least 80;c) at least 90, preferably at least 100, more preferably at least 110,or most preferably at least 120;d) at least 20% of the ratio of semaglutide, preferably at least 50% ofthe ratio of semaglutide, more preferably at least 75% of the ratio ofsemaglutide, even more preferably at least equal to the ratio ofsemaglutide, or most preferably at least twice the ratio of semaglutide;ore) at least equal to the ratio of liraglutide, preferably at least twicethe ratio of liraglutide, more preferably at least three times the ratioof liraglutide, even more preferably at least 5 times the ratio ofliraglutide, or most preferably at least 10 times the ratio ofliraglutide.129. The derivative of any one of embodiments 74-128, for which theGLP-1 receptor binding affinity (IC₅₀) in the presence of 0.005% HSA(low albumin) isa) below 600.00 nM, preferably below 500.00 nM, more preferably below200.00 nM, even more preferably below 100.00 nM, or most preferablybelow 45.00 nM; orb) below 20.00 nM, preferably below 10.00 nM, more preferably below 5.00nM, even more preferably below 2.00 nM, or most preferably below 1.00nM.130. The derivative of any one of embodiments 1-129, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 2.0% HSA (highalbumin) isa) below 900 nM, more preferably below 800 nM, even more preferablybelow 700 nM, or most preferably below 600 nM; orb) below 400.00 nM, preferably below 300.00 nM, more preferably below200.00 nM, even more preferably below 100.00 nM, or most preferablybelow 50.00 nM.131. The derivative of any one of embodiments 1-130, wherein the bindingaffinity to the GLP-1 receptor is measured by way of displacement of¹²⁵I-GLP-1 from the receptor, preferably using a SPA binding assay.132. The derivative of any one of embodiments 1-131, wherein the GLP-1receptor is prepared using a stable, transfected cell line, preferably ahamster cell line, more preferably a baby hamster kidney cell line, suchas BHK tk-ts13.133. The analogue or derivative of any one of embodiments 1-132, whereinthe IC₅₀ value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.134. The derivative of any one of embodiments 74-133, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of liraglutide; and/or higher than that of semaglutide.135. The derivative of embodiment 134, wherein oral bioavailability ismeasured in vivo in rats, as exposure in plasma after direct injectioninto the intestinal lumen.136. The derivative of any one of embodiments 74-135, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is at least 15, preferably at least 30, morepreferably at least 48, still more preferably at least 62, even morepreferably at least 80, or most preferably at least 100.137. The derivative of any one of embodiments 74-136, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is at least 30, preferably at least 40, morepreferably at least 50, still more preferably at least 60, even morepreferably at least 70, or most preferably at least 80.138. The derivative of any one of embodiments 74-137, wherein theDerivative is tested in a concentration of 1000 uM in admixture with 55mg/ml sodium caprate.139. The derivative of any one of embodiments 74-138, wherein maleSprague Dawley rats are used, preferably with a body weight upon arrivalof approximately 240 g.140. The derivative of any one of embodiments 74-139, wherein the ratsare fasted for approximately 18 hours before the experiment.141. The derivative of any one of embodiments 74-140, wherein the ratsare taken into general anaesthesia after having fasted and before theinjection of the derivative in the jejunum.142. The derivative of any one of embodiments 74-141, wherein thederivative is administered in the proximal part of the jejunum (10 cmdistal for the duodenum) or in the mid-intestine (50 cm proximal for thececum).143. The derivative of any one of embodiments 74-142, wherein 100 μl ofthe derivative is injected into the jejunal lumen through a catheterwith a 1 ml syringe, and subsequently 200 μl of air is pushed into thejejunal lumen with another syringe, which is then left connected to thecatheter to prevent flow back into the catheter.144. The derivative of any one of embodiments 74-143, wherein bloodsamples (200 ul) are collected into EDTA tubes from the tail vein atdesired intervals, such as at times 0, 10, 30, 60, 120 and 240 min, andcentrifuged 5 minutes, 10000G, at 4° C. within 20 minutes.145. The derivative of any one of embodiments 74-144, wherein plasma (75ul) is separated, immediately frozen, and kept at −20° C. until analyzedfor plasma concentration of the derivative.146. The derivative of any one of embodiments 74-145, wherein LOCI(Luminescent Oxygen Channeling Immunoassay) is used for analyzing theplasma concentration of the derivative.147. The derivative of any one of embodiments 74-145, which has a moreprotracted profile of action than liraglutide.148. The derivative of embodiment 147, wherein protraction meanshalf-life in vivo in a relevant animal species, such as db/db mice, rat,pig, and/or, preferably, minipig; wherein the derivative is administeredi) s.c., and/or, preferably, ii) s.c.149. The derivative of any one of embodiments 74-148, wherein theterminal half-life (T_(1/2)) after i.v. administration in minipigs isa) at least 12 hours, preferably at least 24 hours, more preferably atleast 36 hours, even more preferably at least 48 hours, or mostpreferably at least 60 hours; orb) at least 0.2 times the half-life of semaglutide, preferably at least0.4 times the half-life of semaglutide, more preferably at least 0.6times the half-life of semaglutide, even more preferably at least 0.8times the half-life of semaglutide, or most preferably at least the sameas the half-life of semaglutide.150. The derivative of embodiment 149, wherein the minipigs are maleGöttingen minipigs.151. The derivative of any one of embodiments 149-150, wherein theminipigs are 7-14 months of age, and preferably weighing from 16-35 kg.152. The derivative of any one of embodiments 149-151, wherein theminipigs are housed individually, and fed once or twice daily,preferably with SDS minipig diet.153. The derivative of any one of embodiments 149-152, wherein thederivative is dosed, i.v., after at least 2 weeks of acclimatisation.154. The derivative of any one of embodiments 149-153, wherein theanimals are fasted for approximately 18 h before dosing and for at least4 h after dosing, and have ad libitum access to water during the wholeperiod.155. The derivative of any one of embodiments 149-154, wherein theDerivative is dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a suitable concentration, preferablyfrom 20-60 nmol/ml.156. The derivative of any one of embodiments 149-155, whereinintravenous injections of the derivative are given in a volumecorresponding to 1-2 nmol/kg.157. The derivative of any one of embodiments 74-156, which is not thecompound of Example 2, preferably not Chem. 31.158. The derivative of any one of embodiments 74-157, which is not thecompound of Examples 7, 8, 9, and 12; preferably not Chem. 36, Chem. 37,Chem. 38, and Chem. 41.158. A peptide according to any one of embodiments 1-73 and 115-127, foruse as a medicament.159. A derivative according to any one of embodiments 74-157, for use asa medicament.160. A peptide according to any one of embodiments 1-73 and 115-127, foruse in the treatment and/or prevention of all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.161. A derivative according to any one of embodiments 74-157, for use inthe treatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.162. Use of a peptide according to any one of embodiments 1-73 and115-127 in the manufacture of a medicament for treatment and/orprevention of all forms of diabetes and related diseases, such as eatingdisorders, cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.163. Use of a derivative according to any one of embodiments 74-157, inthe manufacture of a medicament for treatment and/or prevention of allforms of diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression.164. A method of treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression, by administering a pharmaceutically active amountof a peptide according to any one of embodiments 1-73 and 115-127.165. A method of treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression, by administering a pharmaceutically active amountof a derivative according to any one of embodiments 74-157.166. An intermediate product of the formula Chem. 50 or Chem. 51:

wherein Q represents a bond, or a group of formula Chem. 6:

—(C(R¹⁵)(R¹⁶))_(q)—,  Chem. 6

wherein q is 1-6, and R¹⁵ and R16 independently of each other andindependently for each value of q represent hydrogen, alkyl, carboxyl,or hydroxyl;R represents hydrogen, or alkyl;R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo alkyl,heterocyclyl, or heteroaryl; and each of PG₁ and PG₂ represents aprotection group; with the optional proviso (iii) that R1 and R2 do notboth represent hydrogen;or a pharmaceutically acceptable salt, ester, or amide thereof.167. The intermediate product of embodiment 166, wherein PG₁ is a groupthat reversibly renders the N-atom to which it is attached unreactive,and that can be removed selectively.168. The intermediate product of any one of embodiments 165-167, whereinPG₁ is selected from the group consisting of Boc, Trt, Mtt, Bzl, Tos,Clt, Mmt, Bom, and Fmoc.169. The intermediate product of any one of embodiments 166-168, whereinPG₂ is a group that reversibly renders the —CO group to which it isattached unreactive, and that can be removed selectively.170. The intermediate product of any one of embodiments 165-169, whereinPG₂ is i) —OH, or ii) functionalised as an activated ester.171. The intermediate product of embodiment 170, wherein the activatedester is an ester of p-nitrophenol; 2,4,5-trichlorophenol;N-hydroxysuccinimide; N-hydroxysulfosuccinimide;3,4-dihydro-3-hydroxy-1,2,3-benzotriazine-4-one;5-chloro-8-hydroxyquinoline; N-hydroxy-5-norbornene-2,3-dicarboxylicacid imide; pentafluorophenol; p-sulfotetrafluorophenol;N-hydroxyphthalimide; 1-hydroxybenzotriazole;1-hydroxy-7-azabenzotriazole; N-hydroxymaleimide;4-hydroxy-3-nitrobenzene sulfonic acid; or any other activated esterknown in the art.172. The intermediate product of any one of embodiments 165-171, whereinPG₂ is selected from OPfp, OPnp, and OSuc.173. The intermediate product of any one of embodiments 166-172,wherein, PG₁ is Trt.174. The intermediate product of any one of embodiments 166-173, whereinq is 1-5, preferably 1-4, more preferably 1-3.175. The intermediate product of any one of embodiments 166-174, whereinR15 and R16 independently of each other and independently for each valueof q represent hydrogen.176. The intermediate product of any one of embodiments 166-175, whereinR15 and R16 both represent hydrogen.177. The intermediate product of any one of embodiments 166-176, whereinQ is —(CH₂)_(n), wherein n is 1, 2, or 3.178. The intermediate product of any one of embodiments 166-177, whereinR is hydrogen.179. The intermediate product of any one of embodiments 166-178, whereinR1 and R2 independently represent hydrogen, alkyl, aryl, halogen,hydroxyl, hydroxylalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl,alkoxy, aryloxy, carboxamide, alkyl ester, or aryl ester; whereinpreferably alkyl, hydroxylalkyl, aminoalkyl, carboxylalkyl, alkoxy,and/or alkyl ester contains lower alkyl, straight or branched, morepreferably having 1-6 C-atoms.180. The intermediate product of any one of embodiments 166-179, whereinR1 and R2 independently represent hydrogen, lower alkyl, or loweralkoxy, wherein the lower alkyl and lower alkoxy, independently, have1-5 C-atoms, preferably 1-4 C-atoms, or most preferably 1-3 C-atoms.181. The intermediate product of any one of embodiments 166-180, whereinR1 and R2 independently represent alkyl having 1-2 C-atoms (ethyl, ormethyl), or alkoxy having 1-2 C-atoms (ethoxy, or methoxy).182. The intermediate product of any one of embodiments 166-181, whereinR1 and R2 are methyl or methoxy, preferably methyl.183. The intermediate product of any one of embodiments 166-182, whereinR1 and R2 are both methyl.184. The intermediate product of any one of embodiments 166-183, whereinPG₂ is OH.185. An intermediate product of formula Chem. 50 or Chem. 51:wherein Q represents a bond, or a group of formula Chem. 6:

—(C(R15)(R16))_(q)—,  Chem. 6

wherein q is 1-6, and R15 and R16 independently of each other andindependently for each value of q represent hydrogen, alkyl, carboxyl,or hydroxyl; R represents hydrogen, or alkyl; R1 and R2 independentlyrepresent (i) alkyl, aryl, heterocyclyl, heteroaryl, halogen, hydroxyl,hydroxylalkyl, cyano, amino, aminoalkyl, carboxyl, carboxylalkyl,alkoxy, aryloxy, carboxamide, substituted carboxamide, alkyl ester, arylester, alkyl sulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together formcyclo alkyl, heterocyclyl, or heteroaryl; and each of PG₁ and PG₂represents a protection group, preferably as defined in any one ofembodiments 167-173;or a pharmaceutically acceptable salt, amide, or ester thereof.186. An intermediate compound selected from Chem. 23, Chem. 24, Chem.25, Chem. 26, Chem. 27, Chem. 28, and Chem. 29; or a pharmaceuticallyacceptable salt, amide, or ester thereof.187. The compound of Chem. 40; or a pharmaceutically acceptable salt,amide, or ester thereof.188. A compound characterised by the name of the compound of Example 11herein; or a pharmaceutically acceptable salt, amide, or ester thereof.189. The compound of embodiment 188, which is a compound of embodiment187.190. A peptide intermediate product, which is selected from thefollowing analogues of GLP-1(9-37) (SEQ ID NO: 1): (i) (18K, 22E, 34Q);(ii) (30E, 36K, 38E); (iii) (31H, 34Q); (iv) 34R; (v) (34R, 37K); and(vi) (34R, 37K, 38E);or a pharmaceutically acceptable salt, amide, or ester thereof.191. A peptide intermediate product, which is the following analogue ofGLP-1A(3-37) (SEQ ID NO: 3): (17R, 20R, 33R, 38K); or a pharmaceuticallyacceptable salt, amide, or ester thereof.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods for synthesising andcharacterising peptides and derivatives of the invention. Then follows anumber of examples which relate to the preparation of specific GLP-1peptide derivatives, and at the end a number of examples have beenincluded relating to the activity and properties of these peptides andderivatives (section headed pharmacological methods).

The examples serve to illustrate the invention.

Abbreviations

The following abbreviations are used in the following, in alphabeticalorder:

-   Aib: aminoisobutyric acid (α-aminoisobutyric acid)-   API: Active Pharmaceutical Ingredient-   AUC: Area Under the Curve-   BG: Blood Glucose-   BHK Baby Hamster Kidney-   Boc: t-butyloxycarbonyl-   Bom: benzyloxymethyl-   BW: Body Weight-   Bzl: benzyl-   Clt: 2-chlorotrityl-   collidine: 2,4,6-trimethylpyridine-   cpm: counts per minute-   DCM: dichloromethane-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-   DIC: diisopropylcarbodiimide-   DIPEA: diisopropylethylamine-   DMAP: 4-dimethylaminopyridine-   DMEM: Dulbecco's Modified Eagle's Medium (DMEM)-   EDTA: ethylenediaminetetraacetic acid-   EGTA: ethylene glycol tetraacetic acid-   FCS: Fetal Calf Serum-   Fmoc: 9-fluorenylmethyloxycarbonyl-   HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluoro-phosphate)-   HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate)-   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol-   HOAt: 1-hydroxy-7-azabenzotriazole-   HOBt: 1-hydroxybenzotriazole-   HPLC: High Performance Liquid Chromatography-   HSA: Human Serum Albumin-   IBMX: 3-isobutyl-1-methylxanthine-   Imp: Imidazopropionic acid (also referred to as des-amino histidine,    DesH)-   i.v. intravenously-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl-   IVGTT: Intravenous Glucose Tolerance Test-   LCMS: Liquid Chromatography Mass Spectroscopy-   LYD: Landrace Yorkshire Duroc-   MALDI-MS: See MALDI-TOF MS-   MALDI-TOF MS: Matrix-Assisted Laser Desorption/lonisation Time of    Flight Mass Spectro-scopy-   MeOH: methanol-   Mmt: 4-methoxytrityl-   Mtt: 4-methyltrityl-   NMP: N-methylpyrrolidone-   OBz benzoyl ester-   OEG: 8-amino-3,6-dioxaoctanic acid-   OPfp: pentafluorophenoxy-   OPnp: para-nitrophenoxy-   OSu: O-succinimidyl esters (hydroxysuccinimide esters)-   OSuc: 2,5-dioxo-pyrrolidin-1-yl-   OtBu: tert butyl ester-   PBS: Phosphate Buffered Saline-   PD: Pharmacodynamic-   Pen/Strep: Pencillin/Streptomycin-   PK: Pharmacokinetic-   RP: Reverse Phase-   RP-HPLC: Reverse Phase High Performance Liquid Chromatography-   RT: Room Temperature-   Rt: Retention time-   s.c.: Subcutaneously-   SD: Standard Deviation-   SEC-HPLC: Size Exclusion High Performance Liquid Chromatography-   SPPS: Solid Phase Peptide Synthesis-   tBu: tert. butyl-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane-   TLC: Thin Layer Chromatography-   Tos: tosylate (or para-toluenesulfonyl)-   Tris: tris(hydroxymethyl)aminomethane or    2-amino-2-hydroxymethyl-propane-1,3-diol-   Trt: triphenylmethyl, or trityl-   Trx: tranexamic acid-   UPLC: Ultra Performance Liquid Chromatography

METHODS OF PREPARATION A. General Methods

This section relates to methods for solid phase peptide synthesis (SPPSmethods, including methods for de-protection of amino acids, methods forcleaving the peptide from the resin, and for its purification), as wellas methods for detecting and characterising the resulting peptide (LCMS,MALDI, and UPLC methods). The solid phase synthesis of peptides may insome cases be improved by the use of di-peptides protected on thedi-peptide amide bond with a group that can be cleaved under acidicconditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or2,4,6-trimethoxybenzyl. In cases where a serine or a threonine ispresent in the peptide, pseudoproline di-peptides may be used (availablefrom, e.g., Novabiochem, see also W. R. Sampson (1999), J. Pep. Sci. 5,403). The protected amino acid derivatives used were standard Fmoc-aminoacids (supplied from e.g. Anaspec, IRIS, or Novabiochem). The N-terminalamino acid was Boc protected at the alpha amino group (e.g.Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides with H is at theN-terminus). The epsilon amino group of lysines in the sequence wereeither protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on theroute for attachment of the albumin binding moiety and spacer. Thealbumin binding moiety and/or linker can be attached to the peptideeither by acylation of the resin bound peptide or by acylation insolution of the unprotected peptide. In case of attachment of thealbumin binding moiety and/or linker to the protected peptidyl resin,the attachment can be modular using SPPS and suitably protected buildingblocks such as but not limited to Fmoc-OEG-OH(Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc-tranexamicacid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or4-(9-carboxynonyloxy)benzoic acid tert-butyl ester.

1. Synthesis of Resin Bound Peptide SPPS Method A

SPPS method A refers to the synthesis of a protected peptidyl resinusing Fmoc chemistry on an Applied Biosystems 433 peptide synthesiser(also designated ABI433A synthesiser) in 0.25 mmol or 1.0 mmol scaleusing the manufacturer's FastMoc UV protocols which employ HBTU or HATUmediated couplings in NMP, and UV monitoring of the de-protection of theFmoc protection group.

The starting resin used for the synthesis of peptide amides was asuitable Rink-Amide resin (for peptide amides), or (for peptides with acarboxy C-terminus) either a suitable Wang resin or a suitablechlorotrityl resin. Suitable resins are commercially available from,e.g., Novabiochem.

SPPS Method B

SPPS method B refers to the synthesis of a protected peptidyl resinusing Fmoc chemistry on a microwave-based Liberty peptide synthesiser(CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-loadWang resin available from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wangresin, 0.35 mmol/g). Fmoc-deprotection was with 5% piperidine in NMP atup to 70 or 75° C. The coupling chemistry was DIC/HOAt in NMP. Aminoacid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold) wereadded to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3M amino acid/HOAtsolution were used per coupling for the following scale reactions:Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling timesand temperatures were generally 5 minutes at up to 70 or 75° C. Longercoupling times were used for larger scale reactions, for example 10 min.Histidine amino acids were double coupled at 50° C., or quadruplecoupled if the previous amino acid was sterically hindered (e.g. Aib).Arginine amino acids were coupled at RT for 25 min then heated to 70 or75° C. for 5 min. Some amino acids such as but not limited to Aib, were“double coupled”, meaning that after the first coupling (e.g. 5 min at75° C.), the resin is drained and more reagents are added (amino acid,HOAt and DIC), and the mixture in heated again (e.g. 5 min at 75° C.).When a chemical modification of a lysine side chain was desired, thelysine was incorporated as Lys(Mtt). The Mtt group was removed bywashing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM andNMP. The chemical modification of the lysine was performed either bymanual synthesis (see SPPS method D) or by one or more automated stepson the Liberty peptide synthesiser as described above, using suitablyprotected building blocks (see General methods), optionally including amanual coupling.

SPPS Method D

SPPS method D refers to synthesis of the protected peptidyl resin usingmanual Fmoc chemistry. This was typically used for the attachment of thelinkers and side chains to the peptide backbone. The followingconditions were employed at 0.25 mmol synthesis scale. The couplingchemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess.Coupling conditions were 1-6 h at room temperature. Fmoc-deprotectionwas performed with 20-25% piperidine in NMP (3×20 ml, each 10 min)followed by NMP washings (4×20 mL). Dde- or ivDde-deprotection wasperformed with 2% hydrazine in NMP (2×20 ml, each 10 min) followed byNMP washings (4×20 ml). Mtt- or Mmt-deprotection was performed with 2%TFA and 2-3% TIS in DCM (5×20 ml, each 10 min) followed by DCM (2×20ml), 10% MeOH and 5% DIPEA in DCM (2×20 ml) and NMP (4×20 ml) washings,or by treatment with neat hexafluoroisopropanol (5×20 ml, each 10 min)followed by washings as above. The albumin binding moiety and/or linkercan be attached to the peptide either by acylation of the resin boundpeptide or acylation in solution of the unprotected peptide (see theroutes described below). In case of attachment of the albumin bindingmoiety and/or linker to the protected peptidyl resin the attachment canbe modular using SPPS and suitably protected building blocks (seeGeneral methods).

Attachment to Resin Bound Peptide—Route I:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acidmono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600, 4 molarequivalents relative to resin bound peptide) was dissolved in NMP (25mL), added to the resin and shaken overnight at room temperature. Thereaction mixture was filtered and the resin was washed extensively withNMP, DCM, 2-propanol, methanol and diethyl ether.

Attachment to Resin Bound Peptide—Route II:

The albumin binding moiety was dissolved in NMP/DCM (1:1, 10 ml). Theactivating reagent such as HOBt (4 molar equivalents relative to resin)and DIC (4 molar equivalents relative to resin) was added and thesolution was stirred for 15 min. The solution was added to the resin andDIPEA (4 molar equivalents relative to resin) was added. The resin wasshaken 2 to 24 hours at room temperature. The resin was washed with NMP(2×20 ml), NMP/DCM (1:1, 2×20 ml) and DCM (2×20 ml).

Attachment to Peptide in Solution—Route III:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acidmono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5molar equivalents relative to the peptide was dissolved in an organicsolvent such as acetonitrile, THF, DMF, DMSO or in a mixture ofwater/organic solvent (1-2 ml) and added to a solution of the peptide inwater (10-20 ml) together with 10 molar equivalents of DIPEA. In case ofprotecting groups on the albumin binding residue such as tert-butyl, thereaction mixture was lyophilised overnight and the isolated crudepeptide deprotected afterwards. In case of tert-butyl protection groupsthe deprotection was performed by dissolving the peptide in a mixture oftrifluoroacetic acid, water and triisopropylsilane (90:5:5). After for30 min the mixture was evaporated in vacuo and the crude peptidepurified by preparative HPLC as described later.

SPPS Method E

SPPS method E refers to peptide synthesis by Fmoc chemistry on a PreludeSolid Phase Peptide Synthesiser from Protein Technologies (Tucson, Ariz.85714 U.S.A.). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load fmoc-Lys(Mtt)-Wang resin, 0.35mmol/g). Fmoc-deprotection was with 25% piperidine in NMP for 2×10 min.The coupling chemistry was DIC/HOAt/collidine in NMP. Amino acid/HOAtsolutions (0.3 M in NMP at a molar excess of 3-10 fold) were added tothe resin followed by the same molar equivalent of DIC (3 M in NMP) andcollidine (3 M in NMP). For example, the following amounts of 0.3M aminoacid/HOAt solution were used per coupling for the following scalereactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. Coupling timeswere generally 60 minutes. Some amino acids including, but not limitedto arginine, Aib or histidine were “double coupled”, meaning that afterthe first coupling (e.g. 60 min), the resin is drained and more reagentsare added (amino acid, HOAt, DIC, and collidine), and the mixtureallowed to react gain (e.g. 60 min). Some amino acids and fatty acidderivatives including but not limited to Fmoc-Oeg-OH, Fmoc-Trx-OH,Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were coupled forprolonged time, for example 6 hours. When a chemical modification of alysine side chain was desired, the lysine was incorporated as Lys(Mtt).The Mtt group was removed by washing the resin with DCM and suspendingthe resin in hexafluoroisopropanol/DCM (75:25) for 3×10 minutes followedby washings with DCM, 20% piperidine and NMP. The chemical modificationof the lysine was performed either by manual synthesis (see SPPS methodD) or by one or more automated steps on the Prelude peptide synthesiseras described above using suitably protected building blocks (see Generalmethods).

2. Cleavage of Peptide from the Resin and Purification

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by a 2-3 hour treatment with TFA/TIS/water(95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.The peptide was dissolved in a suitable solvent (such as, e.g., 30%acetic acid) and purified by standard RP-HPLC on a C18, 5 μM column,using acetonitrile/water/TFA. The fractions were analysed by acombination of UPLC, MALDI and LCMS methods, and the appropriatefractions were pooled and lyophilised.

3. Methods for Detection and Characterisation LCMS Methods LCMS Method 1(LCMS1)

An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was usedto identify the mass of the sample after elution from an Agilent 1200series HPLC system. The de-convolution of the protein spectra wascalculated with Agilent's protein confirmation software.

Eluents:

A: 0.1% Trifluoro acetic acid in waterB: 0.1% Trifluoro acetic acid in acetonitrile

Column: Zorbax 5u, 300SB-C3, 4.8×50 mm

Gradient: 25%-95% acetonitrile over 15 min

LCMS Method 2 (LCMS2)

A Perkin Elmer Sciex API 3000 mass spectrometer was used to identify themass of the sample after elution from a Perkin Elmer Series 200 HPLCsystem.

Eluents:

A: 0.05% Trifluoro acetic acid in waterB: 0.05% Trifluoro acetic acid in acetonitrile

Column: Waters Xterra MS C-18×3 mm id 5 μm

Gradient: 5%-90% acetonitrile over 7.5 min at 1.5 ml/min

LCMS Method 3 (LCMS3)

A Waters Micromass ZQ mass spectrometer was used to identify the mass ofthe sample after elution from a Waters Alliance HT HPLC system.

Eluents:

A: 0.1% Trifluoro acetic acid in waterB: 0.1% Trifluoro acetic acid in acetonitrile

Column: Phenomenex, Jupiter C4 50×4.60 mm id 5 μm

Gradient: 10%-90% B over 7.5 min at 1.0 ml/min

LCMS Method 4 (LCMS4)

LCMS4 was performed on a setup consisting of Waters Acquity UPLC systemand LCT Premier XE mass spectrometer from Micromass. The UPLC pump wasconnected to two eluent reservoirs containing:

A: 0.1% Formic acid in waterB: 0.1% Formic acid in acetonitrileThe analysis was performed at RT by injecting an appropriate volume ofthe sample (preferably 2-10 μl) onto the column which was eluted with agradient of A and B.The UPLC conditions, detector settings and mass spectrometer settingswere:Column: Waters Acquity HPLC BEH, C-18, 1.7 μm, 2.1 mm×50 mmGradient: Linear 5%-95% acetonitrile during 4.0 min (alternatively 8.0min) at 0.4 ml/minDetection: 214 nm (analogue output from TUV (Tunable UV detector))MS ionisation mode: API-ESScan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu

HPLC and HPLC Methods Method 05 B5 1

UPLC (method 05_B5_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UBEH130, C18, 130 Å, 1.7 um,2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5) B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 60% A, 40% B to 30% A, 70% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 05 B7 1

UPLC (method 05_B7_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5) B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 80% A, 20% B to 40% A, 60% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 04 A2 1

UPLC (method 04_A2_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 90% A, 10% B to 60% A, 40% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04 A3 1

UPLC (method 04_A3_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 75% A, 25% B to 45% A, 55% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04 A4 1

UPLC (method 04_A4_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonate

B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 65% A, 35% B to 25% A, 65% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 08 B2 1

UPLC (method 08_B2_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 40% A, 60% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 08 B4 1

UPLC (method 08_B4_(—)1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 95% A, 5% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 05 B10 1

UPLC (Method 05_B10_(—)1): The RP-analyses was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5) B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 40% A, 60% B to 20% A, 80% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 02 B4 4

UPLC (Method 02_B4_(—)4): The RP-analysis was performed using a AllianceWaters 2695 system fitted with a Waters 2487 dualband detector. UVdetections at 214 nm and 254 nm were collected using a Symmetry300 C18,5 um, 3.9 mm×150 mm column, 42° C. Eluted with a linear gradient of5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) inwater over 15 minutes at a flow-rate of 1.0 ml/min.

Method 01 B4 1

HPLC (Method 01_B4_(—)1): The RP-analysis was performed using a Waters600S system fitted with a Waters 996 diode array detector. UV detectionswere collected using a Waters 3 mm×150 mm 3.5 um C-18 Symmetry column.The column was heated to 42° C. and eluted with a linear gradient of5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) inwater over 15 minutes at a flow-rate of 1 ml/min.

MALDI-MS Method

Molecular weights were determined using matrix-assisted laser desorptionand ionisation time-of-flight mass spectroscopy, recorded on a Microflexor Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acidwas used.

NMR Method

Proton NMR spectra were recorded using a Brucker Avance DPX 300 (300MHz) with tetramethylsilane as an internal standard. Chemical shifts (6)are given in ppm and splitting patterns are designated as follows: s,singlet; d, doublet; dd, double doublet; dt, double triplet t, triplet,tt, triplet of triplets; q, quartet; quint, quintet; sext, sextet; m,multiplet, and br=broad.

B. Synthesis of Intermediates 1. Synthesis of Mono Esters of FattyDiacids

Overnight reflux of the C12, C14, C16 and C18 diacids withBoc-anhydride, DMAP, and t-butanol in toluene gives predominately thet-butyl mono ester. Obtained is after work-up a mixture of mono acid,diacid and diester. Purification is carried out by washing, short plugsilica filtration and crystallisation.

2. Synthesis of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine

Histamine dihydrochloride (20.47 g; 0.111 mol) and triethylamine (48 mL;0.345 mol) in absolute methanol (400 mL) were stirred at roomtemperature for 10 min. Trifluoroacetic acid ethyl ester (14.6 mL; 0.122mol) in methanol (30 mL) was added dropwise over 30 min at 0° C.Reaction mixture was stirred for 3.5 hrs at room temperature and then itwas evaporated to dryness in vacuo. The residue was dissolved indichlormethane (450 mL) and triethylamine (31 mL; 0.222 mol) was added.Then trityl chloride (34.1 g; 0.122 mol) was added piecewise and mixturewas stirred over night at room temperature. Chloroform (400 mL) andwater (600 mL) were poured into reaction mixture. Aqueous layer wasseparated and extracted with chloroform (3×400 mL). The combined organiclayers were dried over anhydrous magnesium sulfate. Solvent was removedand the beige solid was triturated with hexanes (1000 mL). Suspensionwas filtered to yield2,2,2-trifluoro-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-acetamide aswhite solid.

Yield: 45.54 g (91%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 8.44 (bs, 1H); 7.43 (s, 1H);7.41-7.33 (m, 9H); 7.19-7.10 (m, 6H); 6.65 (s, 1H); 3.66 (q, J=5.9 Hz,2H); 2.79 (t, J=5.9 Hz, 2H).

The above amide (45.54 g; 0.101 mmol) was dissolved in tetrahydrofuran(1000 mL) and methanol (1200 mL). A solution of sodium hydroxide (20.26g; 0.507 mol) in water (500 mL) was added. Mixture was stirred for 2 hrsat room temperature and then it was concentrated in vacuo. The residuewas separated between chloroform (1200 mL) and water (800 mL). Aqueouslayer was extracted with chloroform (3×400 mL). Organic layers werecombined and dried over anhydrous magnesium sulfate. Evaporation of thesolvent yielded brown oil, which was dried for 3 days in vacuo to givethe title product as beige solid.

Yield: 32.23 g (90%).

Overall yield: 82%.

M.p.: 111-113° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.39 (d, J=1.3, 1H); 7.38-7.32(m, 9H); 7.20-7.12 (m, 6H); 6.61 (s, 1H); 3.00 (t, J=6.6 Hz, 2H); 2.70(t, J=6.5 Hz, 2H); 1.93 (bs, 2H).

3. Synthesis of2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid

A mixture of Meldrum's acid (5.52 g, 38.3 mmol), potassium carbonate(26.5 g, 191 mmol) and methyl iodide (7.15 mL, 115 mmol) in acetonitrile(75 mL) was heated at 75° C. in a sealed tube for 7 hrs. The mixture wascooled to room temperature, diluted with dichloromethane (300 mL),filtered and the filtrate evaporated to dryness in vacuo. Ethyl acetate(75 mL), hexanes (75 mL) and water (50 mL) were added and phases wereseparated. The organic layer was washed with 10% aqueous solution ofsodium thiosulfate (50 mL) and water (50 mL); dried over anhydrousmagnesium sulfate and solvent removed in vacuo to give2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione as white solid.

Yield: 6.59 g (79%).

R_(F) (SiO₂, chloroform/ethyl acetate, 98:2): 0.60.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 1.76 (s, 6H); 1.65 (s, 6H).

A solution of 2-(1-Trityl-1H-imidazol-4-yl)-ethyl amine (5.00 g, 14.2mmol) prepared as described above and triethylamine (9.86 mL, 70.7 mmol)in toluene (80 mL) was added dropwise over 50 min to a solution of theabove dione compound (3.65 g, 21.2 mmol) in toluene (40 mL) at 75° C.The mixture was stirred at this temperature for additional 3 hrs (untilthe starting amine was detected on TLC), then it was evaporated todryness. The residue was redissolved in chloroform (300 mL) and washedwith 10% aqueous solution of citric acid (200 mL). The aqueous phase wasextracted with chloroform (2×60 mL); the chloroform phases werecombined, dried over anhydrous magnesium sulfate and solvent removed invacuo. The residue was triturated with hot chloroform (140 mL); hexanes(70 mL) were added and the suspension was stirred at room temperatureovernight. Solids were filtered off, washed with chloroform/hexanesmixture (1:1, 2×50 mL) and dried in vacuo to give the title product.

Yield: 6.73 g (88%).

M.p.: 161-162° C.

R_(F) (SiO₂, chloroform/methanol, 85:15): 0.40.

¹H NMR spectrum (300 MHz, DMSO-d₆, δ_(H)): 12.45 (bs, 1H); 7.66 (t,J=5.1 Hz, 1H); 7.57-7.31 (m, 9H); 7.26 (s, 1H); 7.20-7.02 (m, 6H); 6.66(s, 1H); 3.25 (m, 2H); 2.57 (t, J=7.3 Hz, 2H); 1.21 (s, 6H).

4. Synthesis of 4-(4-tert-Butyl-phenyl)-butyric acid

Aluminum chloride powder (80.0 g, 600 mmol) was added in portions to astirred mixture of tert-butylbenzene (40.0 g, 300 mmol) and succinicanhydride (26.7 g, 267 mmol) and 1,1,2,2-tetrachloroethane (100 mL).After all the aluminum chloride had been added, the mixture was pouredinto a mixture of ice (500 mL) and concentrated hydrochloric acid (100mL). The organic layer was separated, washed with water (500 mL) and thesolvent distilled off. Solid residue was dissolved in hot 15% aqueoussolution of sodium carbonate (1000 mL), filtered, cooled and the acidwas precipitated with hydrochloric acid (acidified to pH=1). The crudeacid was filtered, dried on air and recrystalised from benzene (500 mL)to give 4-(4-tert-butyl-phenyl)-4-oxo-butyric acid as colorlesscrystals.

Yield: 36.00 g (58%).

M.p.: 117-120° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.93 (dm, J=8.3 Hz, 2H); 7.48(dm, J=8.3 Hz, 2H); 3.30 (t, J=6.6 Hz, 2H); 2.81 (t, J=6.6 Hz, 2H); 1.34(s, 9H).

A mixture of the above acid (36.0 g, 154 mmol), potassium hydroxide(25.8 g, 462 mmol), hydrazine hydrate (20 mL, 400 mmol) and ethyleneglycol (135 mL) was refluxed for 3 hrs, and then distilled until thetemperature of the vapor had risen to 196-198° C. After a further 14 hrsreflux, the mixture was allowed to cool slightly, and was then pouredinto cold water (200 mL). The mixture was acidified with concentratedhydrochloric acid (to pH=1) and extracted with dichloromethane (2×400mL). The organic extracts were combined, dried over anhydrous magnesiumsulfate, solvent removed in vacuo and the residue was purified by columnchromatography (Silicagel 60A, 0.060-0.200 mm; eluent: hexanes/ethylacetate 10:1-6:1) to give the title product as off white solid.

Yield: 16.25 g (48%).

M.p.: 59-60° C.

R_(F) (SiO₂, ethyl acetate): 0.60.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.31 (dm, J=8.1 Hz, 2H); 7.12(dm, J=8.1 Hz, 2H); 2.64 (t, J=7.6 Hz, 2H); 2.38 (t, J=7.4 Hz, 2H); 1.96(m, 2H); 1.31 (s, 9H).

5. Synthesis of2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid

Hydroxylamine hydrochloride (15.9 g, 229 mmol) was added to a solutionof 4(5)-imidazolecarboxaldehyde (20.0 g, 209 mmol) and sodium carbonate(12.1 g, 114 mmol) in water (400 mL) and the resulting solution wasstirred at room temperature overnight. The mixture was evaporated to 100mL and cooled in an ice bath. The solids were separated by filtrationand the filtrate was concentrated to 40 mL. After cooling to 0° C.,another portion of crystals was obtained. The solids (23 g) werecombined and recrystallised from ethanol (approx. 160 mL) to affordimidazole-4(5)-carbaldehyde oxime as colorless crystals.

Yield: 15.98 g (69%).

¹H NMR spectrum (300 MHz, acetone-d₃+D₂O, δ_(H)): 7.78 (bs, 1H); 7.74(d, J=0.9 Hz, 1H); 7.43 (s, 1H).

Acetyl chloride (51.0 mL, 718 mmol) was added dropwise to methanol (670mL) at 0° C. under argon. After 30 min, the cooling bath was removed andthe above oxime (16.0 g, 144 mmol) was added, followed by palladium oncarbon (5 wt %, 6.1 g). The mixture was hydrogenated at atmosphericpressure for 17 hrs, then it was filtered through Celite and the solventevaporated to give pure 4-(aminomethyl)-imidazole dihydrochloride ascolorless crystals.

Yield: 23.92 g (98%).

¹H NMR spectrum (300 MHz, D₂O, δ_(H)): 8.72 (s, 1H); 7.60 (s, 1H); 4.33(s, 2H).

The above amine dihydrochloride (18.9 g; 111 mmol) and triethylamine (93mL; 667 mmol) in methanol (1000 mL) were stirred at room temperature for10 min. Trifluoroacetic acid ethyl ester (13.3 mL; 111 mmol) in methanol(30 mL) was added dropwise over 40 min at 0° C. Reaction mixture wasstirred for 18 hrs at room temperature and then it was evaporated todryness in vacuo. The residue was dissolved in dry dichlormethane (2000mL) and triethylamine (31 mL; 222 mmol) was added. Then trityl chloride(31.6 g; 113 mmol) was added and the mixture was stirred overnight atroom temperature. Chloroform (1000 mL) and water (1000 mL) were pouredinto the reaction mixture. Aqueous layer was separated and extractedwith chloroform (2×300 mL). The combined organic layers were dried overanhydrous magnesium sulfate. Solvent was removed and the beige solid wastriturated with hexanes (1000 mL). Suspension was filtered to yield2,2,2-trifluoro-N-(1-trityl-1H-imidazol-4-ylmethyl)-acetamide as whitesolid.

Yield: 46.59 g (96%).

R_(F) (SiO₂, dichloromethane/methanol 95:5): 0.35.

¹H NMR spectrum (300 MHz, DMSO-d₆, δ_(H)): 9.77 (t, J=5.7 Hz, 1H);7.47-7.34 (m, 9H); 7.33 (d, J=1.5 Hz, 1H); 7.13-7.03 (m, 6H); 6.80 (d,J=0.8 Hz, 1H); 4.25 (d, J=5.7 Hz, 2H).

The above amide (46.6 g; 107 mmol) was dissolved in tetrahydrofuran (600mL) and ethanol (310 mL). A solution of sodium hydroxide (21.4 g; 535mmol) in water (85 mL) was added. Mixture was stirred for 5 hrs at roomtemperature and then it was concentrated in vacuo. The residue wasseparated between chloroform (1600 mL) and water (800 mL). Aqueous layerwas extracted with chloroform (4×200 mL). Organic layers were combinedand dried over anhydrous magnesium sulfate. Evaporation of the solventyielded (1-trityl-1H-imidazol-4-yl)-methylamine as off white solid.

Yield: 36.30 g (100%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.38 (d, J=1.3, 1H); 7.36-7.30(m, 9H); 7.18-7.10 (m, 6H); 6.69 (m, 1H); 3.77 (s, 2H); 1.80 (bs, 2H).

A solution of the above amine (10.0 g, 29.5 mmol) and triethylamine(20.5 mL, 147 mmol) in toluene (220 mL) was added dropwise over 45 minto a solution of 2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione (3.65 g,21.2 mmol) in toluene (80 mL) at 75° C. The mixture was stirred at thistemperature for additional 3 hrs (until the starting amine was detectedon TLC), then it was evaporated to dryness. The residue was redissolvedin chloroform (500 mL) and washed with 10% aqueous solution of citricacid (300 mL). The aqueous phase was extracted with chloroform (100 mL);the chloroform phases were combined, washed with water (150 mL) driedover anhydrous magnesium sulfate and solvent removed in vacuo. Theresidue was purified by flash column chromatography (silica gel Fluke60, dichloromethane/methanol 98:2 to 9:1) and crystallised fromchloroform/hexanes mixture to give the title product as beige crystals.

Yield: 9.80 g (73%).

M.p.: 174-175° C.

R_(F) (SiO₂, chloroform/methanol, 85:15): 0.35.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 8.45 (t, J=5.8 Hz, 1H); 7.53(s, 1H); 7.40-7.28 (m, 9H); 7.14-7.01 (m, 6H); 6.84 (s, 1H); 4.39 (d,J=5.8 Hz, 2H); 1.44 (s, 6H).

6. Synthesis of 3-(1-Trityl-1H-imidazol-4-yl)-propyl amine

Ethyl 3-(1-trityl-4-imidazolyl)propionate (93.0 g, 223 mmol) intetrahydrofuran/diethyl ether (1:1, 100 mL) was added dropwise to asuspension of lithium aluminium hydride (17.0 g, 446 mmol) during 1 hr.The mixture was refluxed for 3 hrs, then treated with water (100 mL),20% sodium hydroxide (100 mL) and water (100 mL) under cooling withice/water, filtered and the solid washed with tetrahydrofuran. Theorganic phase was dried over anhydrous potassium carbonate, filtered andevaporated to give 3-(1-trityl-4-imidazolyl)propanol as white solid.

Yield: 68.0 g (82%).

M.p.: 127-129° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.40-7.24 (m, 10H); 7.17-7.06(m, 6H); 6.55 (s, 1H); 3.72 (t, J=5.3 Hz, 2H); 2.68 (t, J=6.6 Hz, 2H);1.86 (m, 2H).

Methanesulfonyl chloride (8 mL, 104 mmol) was added dropwise to asolution of the above alcohol (32.0 g, 86.8 mmol) in dichloromethane(400 mL) and triethyl amine (15.5 mL) at 0° C. during 1 hr. The mixturewas stirred without cooling for an additional 1 hr; then it was washedwith 5% sodium bicarbonate and dried over anhydrous magnesium sulfate.Dichloromethane was evaporated at 30° C. in vacuo and the residual oilymesylate was used directly in the next step.

Yield: 31.2 g (80%).

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.37-7.30 (m, 10H); 7.16-7.09(m, 6H); 6.58 (s, 1 H); 4.24 (t, J=6.3 Hz, 2H); 2.96 (s, 3H); 2.67 (m,2H); 2.10 (m, 2H).

A mixture of the above mesylate (30.0 g, 67 mmol), potassium phtalimide(18.0 g, 100 mmol), sodium iodide (4.0 g, 26.7 mmol) anddimethylformamide (200 mL) was stirred overnight at ambient temperatureand then treated with water (2 L) and benzene (2 L). The organic phasewas dried over anhydrous magnesium sulfate, filtered and solventevaporated giving a residue, which was recrystallised from benzeneyielding 1-trityl-4-(3-phtalimidopropyl)imidazole as white solid.

Yield: 17.2 g (52%).

M.p.: 211-214° C.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.83 (m, 2H); 7.72 (m, 2H);7.39-7.27 (m, 10H); 7.18-7.07 (m, 6H); 6.60 (d, J=0.9 Hz, 1H); 3.72 (t,J=7.4 Hz, 2H); 2.60 (t, J=7.5 Hz, 2H); 1.99 (m, 2H).

The above imidazole derivative (26.6 g, 53.5 mmol) was dissolved inethanol (300 mL) and tetrahydrofuran (150 mL) at 60° C., hydrazinehydrate (50 g, 1 mol) was added and the solution was refluxed for 6 hrsand then heated at 70° C. overnight. The solid was removed by filtrationand the filtrate was treated with 25% aqueous solution of ammonia (2.5l) and dichloromethane (2.5 L). The organic layer was dried overanhydrous potassium carbonate and evaporated to give a residue, whichwas purified by column chromatography on silica gel (Fluke 60,chloroform saturated with ammonia/methanol) giving the title compound aswhite solid.

Yield: 14.2 g (72%).

M.p.: 112-113° C.

R_(F) (SiO₂, chloroform saturated with ammonia/methanol 9:1): 0.30.

¹H NMR spectrum (300 MHz, CDCl₃, δ_(H)): 7.37-7.28 (m, 10H); 7.18-7.09(m, 6H); 6.53 (d, J=1.3 Hz, 1H); 2.74 (t, J=6.9 Hz, 2H); 2.59 (t, J=7.4Hz, 2H); 1.95 (bs, 2H); 1.78 (m, 2H).

7. Synthesis of2,2-Dimethyl-N-[3-(1-trityl-1H-imidazol-4-yl)-propyl]-malonamic acid

2-Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for20 mins and filtrered. Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) wasdissolved i DCM:DMF 1:1 (10 mL) and added to the resin followed by DIPEA(6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL). The resin was shakenovernight at RT. The resin was filtered and washed with DCM:MeOH:DIPEA(17:2:1), DCM, NMP og DCM (2×25 mL of each). The resin was swelled inDMF for 20 mins and filtered. HOAt (3 eq; 9.0 mmol; 1.23 g), DIC (3 eq;9.0 mmol; 1.40 mL) and DMF (25 mL) was added and the resin was shakenfor 90 min at RT. The resin was filtered and3-(1-Trityl-1H-imidazol-4-yl)-propyl amine (1.8 eq; 5.40 mmol; 1.84 g),DIPEA (4 eq; 6.0 mmol; 2.09 mL), and DMF (10 mL) was added. The resinwas shaken for 2 days. The resin was filtered and washed with NMP (5×20mL) and DCM (10×20 mL). 2,2,2-Trifluoroethanol/dichlormethan 1:1 (20 mL)was added to the resin and it was shaked for 2 hrs. The resin was washedwith 2,2,2-Trifluoroethanol/dichlormethan 1:1 (10 mL) and the combinedfiltrates were collected and concentrated in vacuo to yield the titlecompound.

Yield: 600 mg (41%).

LCMS4: m/z=482 (M+1)

UPLC (method 02_B4_(—)4): Rt=8.07 min

¹H NMR spectrum (300 MHz, DMSO-d₆, δ_(H)): 7.36-7.44 (9H, m), 7.07-7.12(6H, m), 6.62 (1H, s), 3.02-3.09 (2H, q), 2.38-2.43 (2H, t), 1.61-1.69(2H, m), 1.26 (6H, s)

8. Synthesis of 2,2-Dimethyl-N-pyridin-2-ylmethylmalonamic acid

Chlorotrityl chloride resin (2.3 g, 3.0 mmol) was swelled in DCM for 20mins and filtered. Dimethylmalonic acid (2 eq; 6.0 mmol; 793 mg) wasdissolved i DCM:NMP 1:1 (10 mL) and added to the resin followed by DIPEA(6 eq; 18.0 mmol; 3.14 mL) and DCM (10 mL). The resin was shakenovernight at RT. The resin was filtered and washed with DCM:MeOH:DIPEA(17:2:1), DCM, NMP og DCM (2×25 mL of each). The resin was swelled inNMP for 20 mins and filtered. HOAt (3 eq; 9.0 mmol; 1.23 g), DIC (3 eq;9.0 mmol; 1.40 mL) and NMP (25 mL) was added and the resin was shakenfor 90 min at RT. The resin was filtrered and 2-(Aminomethyl)pyridine (2eq; 6 mmol; 659 mg), DIPEA (4 eq; 6.0 mmol; 2.09 mL), and NMP (10 mL)was added. The resin was shaken for overnight. The resin was filteredand washed with NMP (5×20 mL) and DCM (10×20 mL). TFA/TIS/water(95:2.5:2.5; 30 mL) was added to the resin and it was shaked for 1 hr,filtered and concentrated in vacuo to yield the title compound.

Yield: 600 mg (41%).

LCMS4: m/z=223 (M+1)

UPLC (method 08_B4_(—)1): Rt=1.79 min

C. Synthesis of Compounds of the Invention Example 1N⁹-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-N^(ε18)-[2-(2-[2-(2-[2-(2-((S)-3-[1-(19-Carboxy-nonadecanoyl)-piperidine-4-carboxyamino]-3-carboxypropionylamino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Lys¹⁸,Glu²²,Gln³⁴]GLP-1(9-37)-peptide

Preparation method: SPPS method E.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acid

LCMS4: m/z=1441.63 (m/3), 1081.62 (m/4)

Example 2N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][His³¹,Gln³⁴]GLP-1(9-37)-peptide

Preparation method: SPPS method B.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid,Fmoc-Oeg-OH, Fmoc-Glu-OtBu, and octadecanedioic acid mono-tert-butylester were coupled using the same coupling condition as an Aib aminoacid.

LCMS4: Rt=2.23 min. m/z=1341 (m/3), 1006 (m/4)

Example 3N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-N^(ε36)-(2-{2-[2-(17-carboxyheptadecanoylamino)ethoxy]ethoxy}acetyl)(Glu³⁰,Lys³⁶)GLP-1(9-37) Glu³⁸-peptide amide

Preparation method: As in Example 2

LCMS4: Rt=6.90 min. m/z=1319 (m/3), 990 (m/4)

Example 4N⁹-{2-[2-(1H-Imidazol-4-yl)-propylcarbamoyl]-2-methyl-propionyl}-N^(ε36)-(2-{2-[2-(17-carboxyheptadecanoylamino)ethoxy]ethoxy}acetyl)[Glu³⁰,Lys³⁶]GLP-1(9-37)Glu³⁸-peptide amide

Preparation method: SPPS method B.2,2-Dimethyl-N-[3-(1-trityl-1H-imidazol-4-yl)-propyl]-malonamic acid,Fmoc-Oeg-OH, and octadecanedioic acid mono-tert-butyl ester were coupledusing the same coupling condition as an Aib amino acid

LCMS4: m/z=1324 (m/3), 993 (m/4)

UPLC (method 04_A3_(—)1): Rt=12.55 min

Example 5N⁹-{2-[2-(1H-Imidazol-4-yl)methylcarbamoyl]-2-methyl-propionyl}-N^(ε36)-(2-{2-[2-(17-carboxyheptadecanoylamino)ethoxy}ethoxy]acetyl)[Glu³⁰,Lys³⁶]GLP-1(9-37)Glu³⁸-peptide amide

Preparation method: SPPS method B.2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid,Fmoc-Oeg-OH, and octadecanedioic acid mono-tert-butyl ester were coupledusing the same coupling condition as an Aib amino acid

LCMS4: m/z=1313 (m/3), 975 (m/4)

UPLC (method 04_A3_(—)1): Rt=12.44 min

Example 6N³-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)-butyrylamino]ethoxy}ethoxy)-acetylamino]ethoxy}-ethoxy)acetyl][Arg¹⁷,Arg²⁰,Arg³³,Lys³⁸]GLP-1A(3-37)-peptide

Preparation method: SPPS method B.2,2-Dimethyl-N-(1-trityl-1H-imidazol-4-ylmethyl)-malonamic acid,Fmoc-Oeg-OH, Fmoc-Glu-OtBu, and octadecanedioic acid mono-tert-butylester were coupled using the same coupling condition as an Aib aminoacid

LCMS4: m/z=1663 (m/3), 1247 (m/4), 998 (m/5)

UPLC (method 04_A3_(—)1): Rt=11.26 min

Example 7N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)-peptide

Preparation method: SPPS method B.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acid.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled usingSPPS method D.

UPLC (method 08_B4_(—)1): Rt=8.81 min.

LCMS4: Rt=2.29 min. m/z=1625 (m/3), 1219 (m/4), 975 (m/5)

Example 8N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-Carboxy-4-[10-(4-carboxy-phenoxy)-decanoylamino]-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)Glu³⁸-peptide

Preparation method: SPPS method B.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acid.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu, and4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared asdescribed in Example 25, step 2 of WO 2006/082204) were coupled usingSPPS method D.

UPLC (method 04_A3_(—)1): Rt=9.32 min.

LCMS4: Rt=2.29 min., m/z=1669 (m/3), 1252 (m/4), 1001 (m/5)

Example 9N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-N^(ε26)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl},N^(ε37)-{2-[2-(2-{2-[2-(2-{(S)-4-[4-(4-tert-Butyl-phenyl)-butyrylamino]-4-carboxy-butyrylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)-peptide

Preparation method: SPPS method B.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as Fmoc-Aib amino acid.8-(9-fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid(commercially available from Iris Biotech), Fmoc-Glu-OtBu and4-(4-t-butylphenyl)butyric acid were coupled using SPPS method D.

UPLC (method 04_A4_(—)1): Rt=10.56 min.

LCMS4: Rt=2.40 min. m/z=940 (m/5), 1174 (m/4), 1565 (m/3)

Example 10N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Arg³⁴]GLP-1(9-37)-peptide

Preparation method: SPPS method E.2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid wascoupled using the same coupling condition as an Aib amino acid

LCMS4: m/z=1367.30 (m/3), 1025.60 (m/4)

Example 11 Comparative Compound N⁹-(5-oxo-5-phenylpentanoyl),N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴]-GLP-1-(9-37)-peptide

Preparation method: SPPS method B

UPLC (method 01_B4_(—)1: Rt=11.48

LCMS2: Rt=5.60 m/z 1356 (m/3)

Example 12 N⁹-[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl],N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴,Lys³]-GLP-1-(9-37)-peptide

Preparation method: SSPS method B.2,2-Dimethyl-N-pyridin-2-ylmethyl-malonamic acid was coupled using thesame coupling condition as used for2,2-Dimethyl-N-[2-(1-trityl-1H-imidazol-4-yl)-ethyl]-malonamic acid inthe previous examples. Fmoc-Glu-OtBu and 4-(9-carboxy-nonyloxy)-benzoicacid tert-butyl ester (prepared as described in Example 25, step 2 of WO2006/082204) were coupled using SPPS method D

UPLC (method 08_B4_(—)1): Rt=8.98 min

LCMS4: Rt=2.23 min. m/z=1624(m/3), 1218 (m/4)

Pharmacological Methods Example 13 In Vitro Potency

The purpose of this example is to test the activity, or potency, of theGLP-1 receptor agonist derivatives in vitro.

The potencies of the GLP-1 receptor agonist derivatives of Examples 1-12were determined as described below, i.e. as the stimulation of theformation of cyclic AMP (cAMP) in a medium containing membranesexpressing the human GLP-1 receptor.

It is noted that the compound of Example 11 (Chem. 40) is a comparativecompound based on compound 215 (p. 24) of WO 2004/067548, whichaccording to FIG. 1 of this WO publication is one of the most potentcompounds of this publication

Principle

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor werestimulated with the GLP-1 receptor agonist derivative in question, andthe potency of cAMP production was measured using the AlphaScreen™ cAMPAssay Kit from Perkin Elmer Life Sciences. The basic principle of TheAlphaScreen Assay is a competition between endogenous cAMP andexogenously added biotin-cAMP. The capture of cAMP is achieved by usinga specific antibody conjugated to acceptor beads.

Cell Culture and Preparation of Membranes

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 5% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the selectionmarker G418.

Cells at approximate 80% confluence were washed 2× with PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and thesupernatant removed. The additional steps were all made on ice. The cellpellet was homogenised by the Ultrathurax for 20-30 sec. in 10 ml ofBuffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at20.000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mMNa-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenised for 20-30sec and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,homogenisation and centrifugation was repeated once and the membraneswere resuspended in Buffer 2. The protein concentration was determinedand the membranes stored at −80° C. until use.

The assay was performed in ½-area 96-well plates, flat bottom (Costarcat. no: 3693). The final volume per well was 50 μl.

Solutions and Reagents

AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No:6760625M); containing Anti-cAMP Acceptor beads (10 U/μl), StreptavidinDonor beads (10 U/μl) and Biotinylated-cAMP (133 U/μl).

AlphaScreen Buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat.no: T3253); 5 mMHEPES (Sigma, cat.no: H3375); 10 mM MgCl₂, 6H₂O (Merck, cat.no: 5833);150 mM NaCl (Sigma, cat.no: S9625); 0.01% Tween (Merck, cat.no: 822184).The following was added to the AlphaScreen Buffer prior to use (finalconcentrations indicated): BSA (Sigma, cat. no. A7906): 0.1%; IBMX(Sigma, cat. no. 15879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM; GTP(Sigma, cat. no. G8877): 1 uM.

cAMP standard (dilution factor in assay=5): cAMP Solution: 5 μL of a 5mM cAMP-stock+495 μL AlphaScreen Buffer.

Suitable dilution series in AlphaScreen Buffer were prepared of the cAMPstandard as well as the GLP-1 analogue or derivative to be tested, e.g.the following eight concentrations of the GLP-1 compound: 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ and 10⁻¹⁴M, and a series from, e.g.,10⁻⁶ to 3×10⁻¹¹ of cAMP.

Membrane/Acceptor Beads

Use hGLP-1/BHK 467-12A membranes; 6 μg/well corresponding to 0.6 mg/ml(the amount of membranes used pr. well may vary)

“No membranes”: Acceptor Beads (15 μg/ml final) in AlphaScreen buffer

“6 μg/well membranes”: membranes+Acceptor Beads (15 μg/ml final) inAlphaScreen buffer

Add 10 μl “No membranes” to the cAMP standard (per well in duplicates)and the positive and negative controls

Add 10 μl “6 μg/well membranes” to GLP-1 and analogues (per well induplicates/triplicates)

Pos. Control: 10 μl “no membranes”+10 μl AlphaScreen Buffer

Neg. Control: 10 μl “no membranes”+10 μl cAMP Stock Solution (50 μM)

As the beads are sensitive to direct light, any handling was in the dark(as dark as possible), or in green light. All dilutions were made onice.

Procedure

1. Make the AlphaScreen Buffer.

2. Dissolve and dilute the GLP-1/Analogues/cAMP standard in AlphaScreenBuffer.3. Make the Donor Beads solution and incubate 30 min. at R.T.4. Add the cAMP/GLP-1/Analogues to the plate: 10 μl per well.5. Prepare membrane/Acceptor Beads solution and add this to the plates:10 μl per well.6. Add the Donor Beads: 30 μl per well.7. Wrap the plate in aluminum foil and incubate on the shaker for 3hours (very slowly) at RT.8. Count on AlphaScreen—each plate pre incubates in the AlphaScreen for3 minutes before counting.

If desired, the fold variation in relation to GLP-1 may be calculated asEC₅₀ (GLP-1)/EC₅₀ (analogue)—3693.2.

Results

The EC₅₀ [pM] values were calculated using the Graph-Pad Prism software(version 5).

All tested derivatives of the invention had a good in vitro potencycorresponding to an EC₅₀ of 2100 pM or below; eight derivatives wereeven more potent having and EC₅₀ at 1000 pM or below; five derivativeshad a still further improved potency corresponding to an EC₅₀ at 500 pMor below; four derivatives were very potent corresponding to an EC₅₀ at300 pM or below; and one derivative had a very good potencycorresponding to an EC₅₀ at 100 pM or below.

The comparative compound of Example 11 was much less potent, namely withan EC₅₀ of above 8000 pM.

Example 14 GLP-1 Receptor Binding

The purpose of this experiment is to investigate the binding to theGLP-1 receptor of the GLP-1 agonist derivatives, and how the binding ispotentially influenced by the presence of albumin. This is done in an invitro experiment as described below.

The binding affinity of the GLP-1 receptor agonist derivatives ofExamples 1-12 to the human GLP-1 receptor was measured by way of theirability to displace of ¹²⁵I-GLP-1 from the receptor.

It is noted that the compound of Example 11 (Chem. 40) is a comparativecompound based on compound 215 (p. 24) of WO 2004/067548, whichaccording to FIG. 1 of this WO publication is one of the most potentcompounds of this publication.

In order to test the binding of the derivatives to albumin, the assaywas performed with a low concentration of albumin (0.005%—correspondingto the residual amount thereof in the tracer), as well as with a highconcentration of albumin (2.0% added).

A shift in the binding affinity, IC₅₀, is an indication that the peptidein question binds to albumin, and thereby a prediction of a potentialprotracted pharmacokinetic profile of the peptide in question in animalmodels.

Conditions

Species (in vitro): Hamster

Biological End Point: Receptor Binding

Assay Method: SPA

Receptor: GLP-1 receptor

Cell Line: BHK tk-ts13

Cell Culture and Membrane Purification

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 10% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418.

The cells (approx. 80% confluence) were washed twice in PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), following which they were separated bycentrifugation at 1000 rpm for 5 min. The cells/cell pellet must be kepton ice to the extent possible in the subsequent steps. The cell pelletwas homogenised with Ultrathurrax for 20-30 seconds in a suitable amountof Buffer 1 (depending on the amount of cells, but e.g. 10 ml). Thehomogenate was centrifuged at 20000 rpm for 15 minutes. The pellet wasresuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. Thisstep was repeated once more. The resulting pellet was resuspended inBuffer 2, and the protein concentration was determined. The membraneswere stored at minus 80° C.

Buffer 1: 20 mM Na-HEPES+10 mM EDTA, pH 7.4

Buffer 2: 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4

Binding Assay:

SPA:

Test compounds, membranes, SPA-particles and [¹²⁵I]-GLP-1(7-36)NH₂ werediluted in assay buffer. 25 ul (micro liter) of test compounds wereadded to Optiplate. HSA (“high albumin” experiment containing 2% HSA),or buffer (“low albumin” experiment containing 0.005% HSA), was added(50 ul). 5-10 ug protein/sample was added (50 ul) corresponding to0.1-0.2 mg protein/ml (to be preferably optimised for each membranepreparation). SPA-particles (Wheatgerm agglutinin SPA beads, PerkinElmer, #RPNQ0001) were added in an amount of 0.5 mg/well (50 ul). Theincubation was started with [¹²⁵I]-GLP-1]-(7-36)NH₂ (final concentration0.06 nM corresponding to 49.880 DPM, 25 ul). The plates were sealed withPlateSealer and incubated for 120 minutes at 30° C. while shaking. Theplates were centrifuged (1500 rpm, 10 min) and counted in Topcounter.

Assay buffer:

50 mM HEPES

5 mM EGTA

5 mM MgC12

0.005% Tween 20

pH 7.4

HSA was SIGMA A1653

Calculations

The IC₅₀ value was read from the curve as the concentration whichdisplaces 50% of ¹²⁵I-GLP-1 from the receptor, and the ratio of[(IC₅₀/nM) high HSA]/[(IC₅₀/nM) low HSA] was determined.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 receptor agonist derivatives also bindto albumin. This is a generally desirable effect, which extends theirlifetime in plasma. Therefore, the IC₅₀ value at high albumin willgenerally be higher than the IC₅₀ value at low albumin, corresponding toa reduced binding to the GLP-1 receptor, caused by albumin bindingcompeting with the binding to the GLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low).

Results

The following results were obtained, where “ratio” refers to [(IC₅₀/nM)high HSA]/[(IC₅₀/nM) low HSA]):

All derivatives had a ratio above 10; ten were above 20; six derivativeswere above 30; four derivatives were above 50; and one derivative wasabove 100. The comparative compound of Example 11, had a ratio above300.

Furthermore as regards IC₅₀ (low albumin), all derivatives, except thecomparative compound of Example 11, had an IC₅₀ (low albumin) below 40nM; all but one below 20 nM; all but four were below 10.0 nM; five werebelow 5.00 nM; and three derivatives were below 1.00 nM.

Finally as regards IC₅₀ (high albumin), all derivatives of the inventionhad an IC₅₀ (high albumin) below 900.00 nM; nine were below 500.00 nM;four were below 100.00 nM; and two derivatives were below 50.00 nM. TheIC₅₀ (high albumin) for the comparative compound of Example 11 was above800.00 nM.

Example 15 Estimate of Oral Bioavailability

The purpose of this experiment is to estimate the oral bioavailabilityof the GLP-1 receptor agonist derivatives.

To this end, the exposure in plasma after direct injection into theintestinal lumen of the compounds is studied in vivo in rats, asdescribed in the following.

The compounds are tested in a concentration of 1000 uM in a solution of55 mg/ml sodium caprate.

32 male Sprague Dawley rats with a body weight upon arrival ofapproximately 240 g are obtained from Taconic (Denmark) and assigned tothe different treatments by simple randomisation, 4 rats per group. Therats are fasted for approximately 18 hours before the experiment andtaken into general anaesthesia (Hypnorm/Dormicum).

The compounds are administered in the jejunum either in the proximalpart (10 cm distal for the duodenum) or in the mid-intestine (50 cmproximal for the cecum). A PE50-catheter, 10 cm long is inserted intothe jejunum, forwarded at least 1.5 cm into the jejunum, and securedbefore dosing by ligature around the gut and the catheter with 3/0suture distal to tip to prevent leak or catheter displacement. Catheteris placed without syringe and needle and 2 ml saline is administeredinto abdomen before closing the incision with wound clips.

100 μl of the respective compound is injected into the jejunal lumenthrough the catheter with a 1 ml syringe. Subsequently, 200 μl of air ispushed into the jejunal lumen with another syringe to “flush” thecatheter. This syringe is leaved connected to the catheter to preventflow back into the catheter.

Blood samples (200 ul) are collected at desired intervals (usually attimes 0, 10, 30, 60, 120 and 240 min) into EDTA tubes from the tail veinand centrifuged 5 minutes, 10000G, at 4° C. within 20 minutes. Plasma(75 ul) is separated to Micronic tubes, immediately frozen, and kept at−20° C. until analyzed for plasma concentration of the respective GLP-1receptor agonist derivative with LOCI (Luminescent Oxygen ChannelingImmunoassay), generally as described for the determination of insulin byPoulsen and Jensen in Journal of Biomolecular Screening 2007, vol. 12,p. 240-247. The donor beads are coated with streptavidin, while acceptorbeads are conjugated with a monoclonal antibody recognising amid-/C-terminal epitope of the peptide. Another monoclonal antibody,specific for the N-terminus, is biotinylated. The three reactants arecombined with the analyte and formed a two-sited immuno-complex.Illumination of the complex released singlet oxygen atoms from the donorbeads, which are channeled into the acceptor beads and triggeredchemiluminescence which is measured in an Envision plate reader. Theamount of light is proportional to the concentration of the compound.

After the blood sampling the rats are sacrificed under anaesthesia andthe abdomen is opened to verify correct catheter placement.

The mean (n=4) plasma concentrations (pmol/l) are determined as afunction of time. The ratio of plasma concentration (pmol/l) divided bythe concentration of the dosing solution (μmol/l) is calculated for eachtreatment, and the results for t=30 min (30 minutes after the injectionof the compound in the jejunum) are assessed (dose-corrected exposure at30 min) as a surrogate measure of intestinal bioavailability. Thedose-corrected exposure has been shown to correlate significantly withthe actual bioavailability.

The results may be given as dose-corrected exposure at 30 min whichrefers to (the plasma concentration 30 minutes after injection of thecompound in the jejunum (pM)), divided by (the concentration of thecompound in the dosing solution (pM)).

Example 16 Pharmacokinetics in Minipigs

The purpose of this study is to determine the protraction in vivo of theGLP-1 receptor agonist derivatives after i.v. administration tominipigs, i.e. the prolongation of their time of action. This is done ina pharmacokinetic (PK) study, where the terminal half-life of thederivative in question is determined. By terminal half-life is generallymeant the period of time it takes to halve a certain plasmaconcentration, measured after the initial distribution phase.

Male Göttingen minipigs obtained from Ellegaard Göttingen Minipigs(Dalmose, Denmark) approximately 7-14 months of age and weighing fromapproximately 16-35 kg are used in the studies. The minipigs are housedindividually and fed restrictedly once or twice daily with SDS minipigdiet (Special Diets Services, Essex, UK). After at least 2 weeks ofacclimatisation two permanent central venous catheters are implanted invena cava caudalis or cranialis in each animal. The animals are allowed1 week recovery after the surgery, and are then used for repeatedpharmacokinetic studies with a suitable wash-out period between dosings.

The animals are fasted for approximately 18 h before dosing and for atleast 4 h after dosing, but had ad libitum access to water during thewhole period.

The compounds are dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a concentration of usually from20-60 nmol/ml. Intravenous injections (the volume corresponding tousually 1-2 nmol/kg, for example 0.033 ml/kg) of the compounds are giventhrough a catheter, and blood is sampled at predefined time points forup till 13 days post dosing (preferably through the other catheter).Blood samples (for example 0.8 ml) are collected in EDTA buffer (8 mM)and then centrifuged at 4° C. and 1942G for 10 minutes. Plasma ispippetted into Micronic tubes on dry ice, and kept at −20° C. untilanalyzed for plasma concentration of the respective GLP-1 compound usingELISA or a similar antibody based assay or LC-MS. Individual plasmaconcentration-time profiles are analyzed by a non-compartmental model inWinNonlin v. 5.0 (Pharsight Inc., Mountain View, Calif., USA), and theresulting terminal half-lives (harmonic mean) determined.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A GLP-1 receptor agonist peptide having the formula Chem. 1:Y—Z—P,  Chem 1 wherein P represents a fragment of a GLP-1 receptoragonist peptide lacking the two N-terminal amino acid residues; Zrepresents a group of the formula Chem. 2:

wherein W represents a group of formula Chem. 3:

wherein R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or R1 and R2 together form (ii) cyclo alkyl,heterocyclyl, or heteroaryl, with the proviso that (iii) R1 and R2 donot both represent hydrogen; and Y represents a group of formula Chem. 4or Chem. 5:

wherein X₁ is N, O, or S; X₂, X₃, X₄, and X₅ independently represent C,or N, with the proviso that at least one of X₂, X₃, X₄ and X₅ is C; R11,R12, R13, and R14 independently represent hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl; Q represents a bond, or a group of formulaChem. 6:*—(C(R15)(R16))_(q)—*  Chem. 6 wherein q is 1-6, and R15 and R16independently of each other and independently for each value of qrepresent hydrogen, alkyl, carboxyl, or hydroxyl; and R representshydrogen, or alkyl; or a pharmaceutically acceptable salt, amide, orester thereof.
 2. A GLP-1 receptor agonist peptide selected from thefollowing: (i)N⁹-[-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Lys¹⁸,Glu²²,Gln³⁴]GLP-1(9-37)-peptide;(ii)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[His³¹,Gln³⁴]GLP-1(9-37)-peptide;(iii)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)-Glu³⁸-peptideamide; (iv)N⁹-{2-[2-(1H-Imidazol-4-yl)propylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)Glu³⁸-peptideamide; (v)N⁹-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Glu³⁰,Lys³⁶]GLP-1(9-37)-Glu³⁸-peptideamide; (vi)N³-{2-[2-(1H-Imidazol-4-yl)-methylcarbamoyl]-2-methyl-propionyl}-[Arg¹⁷,Arg²⁰,Arg³³,Lys³⁸]GLP-1A(3-37)-peptide;(vii)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg³⁴,Lys³⁷]GLP-1(9-37)-peptide;(iix)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg³⁴,Lys³⁷]GLP-1(9-37)Glu³⁸-peptide;(ix)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}-[Arg³⁴,Lys³⁷]GLP-1(9-37)-peptide; (x)N⁹-{2-[2-(1H-Imidazol-4-yl)-ethylcarbamoyl]-2-methyl-propionyl}[Arg³⁴]GLP-1(9-37)-peptide;and (xii)N⁹-[2,2-dimethyl-3-oxo-3-(pyridin-2-ylmethylamino)propanoyl]-[Arg³⁴,Lys³⁷]-GLP-1-(9-37)-peptide;or a pharmaceutically acceptable salt, amide, or ester thereof.
 3. Aderivative of a peptide of claim 1, or a pharmaceutically acceptablesalt, amide, or ester thereof.
 4. The derivative of claim 3 which has analbumin binding moiety attached to a lysine residue of the peptide. 5.The derivative of claim 4, in which the albumin binding moiety comprisesa protracting moiety selected from Chem. 8, Chem. 9, and Chem. 10:HOOC—(CH₂)_(x)—CO—*  Chem. 8HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 9R¹⁸—C₆H₄—(CH₂)_(z)—CO—*  Chem. 10 in which x is an integer in the rangeof 6-18, y is an integer in the range of 3-17, z is an integer in therange of 1-5, and R¹⁸ is a group having a molar mass not higher than 150Da.
 6. The derivative of claim 5, wherein the albumin binding moietyfurther comprises a linker selected from Chem. 11, Chem. 12, Chem. 13,and Chem. 14:*—NH—CH₂—CH₂—(O—CH₂—CH₂)_(k)—O—(CH₂)_(n)—CO—*  Chem. 11*—NH—C(COOH)—(CH₂)₂—CO—*  Chem. 12*—N—C((CH₂)₂COOH)—CO—*  Chem. 13*—NC₅H₈—CO—*  Chem. 14 wherein k is an integer in the range of 1-5, andn is an integer in the range of 1-5.
 7. A derivative of a GLP-1 receptoragonist peptide selected from the following: Chem. 30, Chem. 31, Chem.32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem.39, and Chem. 41; or a pharmaceutically acceptable salt, amide, or esterthereof.
 8. An intermediate compound of the formula Chem. 50 or Chem.51:

wherein Q represents a bond, or a group of formula Chem. 6:—(C(R¹⁵)(R¹⁶))_(q)—,  Chem. 6 wherein q is 1-6, and R¹⁵ and R16independently of each other and independently for each value of qrepresent hydrogen, alkyl, carboxyl, or hydroxyl; R represents hydrogen,or alkyl; R1 and R2 independently represent (i) hydrogen, alkyl, aryl,heterocyclyl, heteroaryl, halogen, hydroxyl, hydroxylalkyl, cyano,amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy,carboxamide, substituted carboxamide, alkyl ester, aryl ester, alkylsulfonyl, or aryl sulfonyl, or (ii) R1 and R2 together form cyclo alkyl,heterocyclyl, or heteroaryl; and each of PG₁ and PG₂ represents aprotection group; or a pharmaceutically acceptable salt, ester, or amidethereof.
 9. An intermediate compound selected from Chem. 23, Chem. 24,Chem. 25, Chem. 26, Chem. 27, Chem. 28, and Chem. 29; or apharmaceutically acceptable salt, amide, or ester thereof, where


10. The compound of Chem. 40: N⁹-(5-oxo-5-phenylpentanoyl),N^(ε26)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg³⁴]-GLP-1-(9-37)-peptide;or a pharmaceutically acceptable salt, amide, or ester thereof.
 11. Apeptide intermediate product, which is selected from a) the followinganalogues of GLP-1(9-37) (SEQ ID NO: 1): (i) (18K, 22E, 34Q); (ii) (30E,36K, 38E); (iii) (31H, 34Q); (iv) 34R; (v) (34R, 37K); and (vi) (34R,37K, 38E); and b) the following analogue of GLP-1A(3-37) (SEQ ID NO: 3):(17R, 20R, 33R, 38K); or a pharmaceutically acceptable salt, amide, orester thereof 12-15. (canceled)
 16. A derivative of a peptide of claim2, or a pharmaceutically acceptable salt, amide, or ester thereof. 17.The derivative of claim 16 which has an albumin binding moiety attachedto a lysine residue of the peptide.
 18. The derivative of claim 17, inwhich the albumin binding moiety comprises a protracting moiety selectedfrom Chem. 8, Chem. 9, and Chem. 10:HOOC—(CH₂)_(x)—CO—*  Chem. 8HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 9R¹⁸—C₆H₄—(CH₂)_(z)—CO—*  Chem. 10 in which x is an integer in the rangeof 6-18, y is an integer in the range of 3-17, z is an integer in therange of 1-5, and R¹⁸ is a group having a molar mass not higher than 150Da.
 19. The derivative of claim 18, wherein the albumin binding moietyfurther comprises a linker selected from Chem. 11, Chem. 12, Chem. 13,and Chem. 14:*—NH—CH₂—CH₂—(O—CH₂—CH₂)_(k)—O—(CH₂)_(n)—CO—*  Chem. 11*—NH—C(COOH)—(CH₂)₂—CO—*  Chem. 12*—N—C((CH₂)₂COOH)—CO—*  Chem. 13*—NC₅H₈—CO—*  Chem. 14 wherein k is an integer in the range of 1-5, andn is an integer in the range of 1-5.
 20. A pharmaceutical compositioncomprising a GLP-1 receptor agonist peptide of claim 1 and apharmaceutically acceptable carrier or diluent.
 21. A method of treatingdiabetes in a subject in need of such treatment, said method comprisingadministering to said subject an effective amount of the pharmaceuticalcomposition of claim
 20. 22. A pharmaceutical composition comprising aGLP-1 receptor agonist peptide of claim 2 and a pharmaceuticallyacceptable carrier or diluent.
 23. A method of treating diabetes in asubject in need of such treatment, said method comprising administeringto said subject an effective amount of the pharmaceutical composition ofclaim
 22. 24. The derivative of claim 4, wherein the albumin bindingmoiety further comprises a linker selected from the group consisting of: